JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Medicine

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

Published: June 26th, 2016

DOI:

10.3791/54306

1Department of Immunotherapeutics and Biotechnology, School of Pharmacy, Texas Tech University Health Science Center, 2Merck Research Labs, 3Department of Surgery, Memorial Sloan Kettering Cancer Center

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

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 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. Despi....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 "Guide for the Care and Use of Laboratory Animals" 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.

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
4T1 cell line  American Type Culture Collection, Manassas, VA, USA CRL 2539 Tumor cells
4T1-GFP cell line Caliper life sciences/ Perkin Elmer, Waltham, MA, USA BW128090 Tumor cells
RPMI Corning, Corning, NY, USA 10-040-CM Media
Heat inactivated FBS Gibco (Thermo Scientific), USA 10082147 Media
Penicillin Streptomycin Fisher Scientific, Waltham, MA, USA MT-300-02-CI Media
PBS Fisher Scientific, Waltham, MA, USA BP399-20 Dilute with distilled water
Trypsin 0.25% with EDTA Hyclone, Logan, Utah, USA SH30042.02 Tissue culture supplies
T75 cm2 flask Fisher Scientific, Waltham, MA, USA 12-565-32 Tissue culture supplies
15ml conical tube BD falcon, Franklin Lakes, NJ, USA 352096 Tissue culture supplies
50ml conical tube BD falcon, Franklin Lakes, NJ, USA 352098 Tissue culture supplies
60mm2 Petri dish Fisher Scientific, Waltham, MA, USA AS4052 For lung imaging 
Isoflurane (Isothesia) Butler Schein Animal health, Dublin, OH, USA NDC 11695-6776-2 Mouse anesthesia
Clodronate liposomes Formumax Scientific Inc, Palo Alto, CA, USA F70101C-N Macrophages depletion
Control liposomes Formumax Scientific Inc, Palo Alto, CA, USA F70101-N Control PBS-liposomes
29 gauge insulin syringes (12.7 mm and 0.5 ml capacity)- Reli-On Walmart, Bentonville, AR, USA For tumor cell injection
Hair removal cream (Nair) Walmart, Bentonville, AR, USA
Paraformaldehyde solution (4%) Affymetrix, Santa Clara, CA, USA 19943-I Lt Dilute to 4% or 1% using 1X PBS
OCT compound Fisher Scientific, Waltham, MA, USA 230-730-571 For freezing tissue in cryomolds
Fluoro-Gel-II with DAPI Electron Microscopy Sciences, Hatfield, PA, USA 17985-51 Mounting medium
Sucrose Sigma, St. Louis, MO, USA S-9378 Cryopreservation
Collagenase P Roche, Basel, Switzerland 11249002001 Components of tissue digestion buffer
Dnase I Roche, Basel, Switzerland 10104159001 Components of tissue digestion buffer
Trypsin inhibitor Sigma, St. Louis, MO, USA T9253 Components of tissue digestion buffer
40 micron cell strainers Fisher Scientific, Waltham, MA, USA 22-363-547 Used in tissue digestion to remove clumps
Trustain FcX-Fc Block (CD16/CD32) Biolegend, San Diego, CA, USA 101320 Antibodies for flow cytometry
BV605 CD45 Biolegend, San Diego, CA, USA 103139 Antibodies for flow cytometry
PE CD11b Biolegend, San Diego, CA, USA 101207 Antibodies for flow cytometry
PE Cy7 F4/80  Biolegend, San Diego, CA, USA 123113 Antibodies for flow cytometry
APC/Cy7 CD11c Biolegend, San Diego, CA, USA 117323 Antibodies for flow cytometry
PerCPcy5.5 IA/IE (MHCII)  Biolegend, San Diego, CA, USA 107625 Antibodies for flow cytometry
PE CD80 Biolegend, San Diego, CA, USA 104707 Antibodies for flow cytometry
AF647 CD86 Biolegend, San Diego, CA, USA 105019 Antibodies for flow cytometry
Fixable viability Dye eflour 506 eBioscience, San Diego, CA,USA 65-0866 Antibodies for flow cytometry
Cryostat Leica Biosystems, Buffalo Grove, IL, USA CM1850 Cryosectioning
UVP iBox Explorer UVP Inc, Upland, CA, USA Mouse and lung fluorescent imaging
Aperio Scanscope CS Leica Biosystems, Buffalo Grove, IL, USA Digital pathology
BD LSRFortessa  BD Biosciences, Franklin Lakes, NJ, USA Flow cytometry/data acquisition
Nikon A1 confocal TE2000 microscope Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A. Imaging and quantifying GFP fluorescence in lung cryosections
UVP visionworks software (Version 7.1RC3.38) UVP Inc, Upland, CA, USA Imaging software for iBOX
Aperio Imagescope software (v12.1.0.5029)  Leica Biosystems, Buffalo Grove, IL, USA Imaging software for analysis of digital slides
Flow JO software (version 9.8.1) Flow JO LLC, Ashland, OR, USA Analysis of flow cytometric data
NIS Elements AR (version 4.20.01) 64 Bit Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A. Acquisition and analysis of lung cryosections for GFP 

  1. Sceneay, J., Smyth, M. J., Moller, A. The pre-metastatic niche: finding common ground. Cancer Metastasis Rev. , (2013).
  2. Fidler, I. J. The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited. Nat Rev Cancer. 3, 453-458 (2003).
  3. Kaplan, R. N., et al. VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature. 438, 820-827 (2005).
  4. Hiratsuka, S., et al. MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell. 2, 289-300 (2002).
  5. Hiratsuka, S., et al. The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase. Nat Cell Biol. 10, 1349-1355 (2008).
  6. Yan, H. H., et al. Gr-1+CD11b+ myeloid cells tip the balance of immune protection to tumor promotion in the premetastatic lung. Cancer Res. 70, 6139-6149 (2010).
  7. Gao, D., et al. Myeloid progenitor cells in the premetastatic lung promote metastases by inducing mesenchymal to epithelial transition. Cancer Res. 72, 1384-1394 (2012).
  8. Davies, L. C., Jenkins, S. J., Allen, J. E., Taylor, P. R. Tissue-resident macrophages. Nat Immunol. 14, 986-995 (2013).
  9. Holt, P. G., Strickland, D. H., Wikstrom, M. E., Jahnsen, F. L. Regulation of immunological homeostasis in the respiratory tract. Nat Rev Immunol. 8, 142-152 (2008).
  10. Sharma, S. K., et al. Pulmonary alveolar macrophages contribute to the premetastatic niche by suppressing antitumor T cell responses in the lungs. J. Immunol. 194, 5529-5538 (2015).
  11. Pulaski, B. A., Ostrand-Rosenberg, S., Coligan, J. E. Mouse 4T1 breast tumor model. Current protocols in immunology. , (2001).
  12. Gupta, G. P., Massague, J. Cancer metastasis: building a framework. Cell. 127, 679-695 (2006).
  13. Buiting, A. M., Van Rooijen, N. Liposome mediated depletion of macrophages: an approach for fundamental studies. J. Drug Target. 2, 357-362 (1994).
  14. Vadrevu, S. K., et al. Complement c5a receptor facilitates cancer metastasis by altering T-cell responses in the metastatic niche. Cancer Res. 74, 3454-3465 (2014).
  15. Walrath, J. C., Hawes, J. J., Van Dyke, T., Reilly, K. M. Genetically engineered mouse models in cancer research. Adv. Cancer Res. 106, 113-164 (2010).
  16. Bosiljcic, M., et al. Myeloid suppressor cells regulate the lung environment--letter. Cancer Res. 71, 5050-5051 (2011).
  17. Yan, H. H., et al. Myeloid Suppressor Cells Regulate the Lung Environment-Response. Cancer Res. 71, 5052-5053 (2011).
  18. Van Rooijen, N., Sanders, A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J. Immunol. Methods. 174, 83-93 (1994).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved