Clinical & Experimental Metastasis

, Volume 25, Issue 8, pp 877–885 | Cite as

Establishment and quantitative imaging of a 3D lung organotypic model of mammary tumor outgrowth

  • Michelle D. Martin
  • Barbara Fingleton
  • Conor C. Lynch
  • Sam Wells
  • J. Oliver McIntyre
  • David W. Piston
  • Lynn M. MatrisianEmail author


The lung is the second most common site of metastatic spread in breast cancer and experimental evidence has been provided in many systems for the importance of an organ-specific microenvironment in the development of metastasis. To better understand the interaction between tumor and host cells in this important secondary site, we have developed a 3D in vitro organotypic model of breast tumor metastatic growth in the lung. In our model, cells isolated from mouse lungs are placed in a collagen sponge to serve as a scaffold and co-cultured with a green fluorescent protein-labeled polyoma virus middle T antigen (PyVT) mammary tumor cell line. Analysis of the co-culture system was performed using flow cytometry to determine the relative constitution of the co-cultures over time. This analysis determined that the cultures consisted of viable lung and breast cancer cells over a 5-day period. Confocal microscopy was then used to perform live cell imaging of the co-cultures over time. Our studies determined that host lung cells influence the ability of tumor cells to grow, as the presence of lung parenchyma positively affected the proliferation of the mammary tumor cells in culture. In summary, we have developed a novel in vitro model of breast tumor cells in a common metastatic site that can be used to study tumor/host interactions in an important microenvironment.


Breast cancer Metastasis Microenvironment Organotypic co-culture Host–tumor interactions 



Green fluorescent protein


Polyoma virus middle T antigen


Phosphate buffered saline


Hematoxylin and eosin


Fetal calf serum







This work was supported by a grant from the NIH (R01-CA84360 to LMM). We are grateful to Dr. Carlos Arteaga and Dr. Shimian Qu for the pMSCV-GFP vector.


  1. 1.
    Hiratsuka S, Nakamura K, Iwai S et al (2002) MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell 2(4):289–300. doi: 10.1016/S1535-6108(02)00153-8 PubMedCrossRefGoogle Scholar
  2. 2.
    Itoh T, Tanioka M, Matsuda H et al (1999) Experimental metastasis is suppressed in MMP-9-deficient mice. Clin Exp Metastasis 17(2):177–181. doi: 10.1023/A:1006603723759 PubMedCrossRefGoogle Scholar
  3. 3.
    Muller A, Homey B, Soto H et al (2001) Involvement of chemokine receptors in breast cancer metastasis. Nature 410(6824):50–56. doi: 10.1038/35065016 PubMedCrossRefGoogle Scholar
  4. 4.
    Gupta GP, Nguyen DX, Chiang AC et al (2007) Mediators of vascular remodelling co-opted for sequential steps in lung metastasis. Nature 446(7137):765–770. doi: 10.1038/nature05760 PubMedCrossRefGoogle Scholar
  5. 5.
    Debnath J, Mills KR, Collins NL et al (2002) The role of apoptosis in creating and maintaining luminal space within normal and oncogene-expressing mammary acini. Cell 111(1):29–40. doi: 10.1016/S0092-8674(02)01001-2 PubMedCrossRefGoogle Scholar
  6. 6.
    Streuli CH, Bissell MJ (1990) Expression of extracellular matrix components is regulated by substratum. J Cell Biol 110:1405–1415. doi: 10.1083/jcb.110.4.1405 PubMedCrossRefGoogle Scholar
  7. 7.
    Debnath J, Muthuswamy SK, Brugge JS (2003) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30(3):256–268. doi: 10.1016/S1046-2023(03)00032-X PubMedCrossRefGoogle Scholar
  8. 8.
    Kim JB (2005) Three-dimensional tissue culture models in cancer biology. Semin Cancer Biol 15(5):365–377. doi: 10.1016/j.semcancer.2005.05.002 PubMedCrossRefGoogle Scholar
  9. 9.
    Kim JB, Stein R, O’Hare MJ (2004) Three-dimensional in vitro tissue culture models of breast cancer—a review. Breast Cancer Res Treat 85(3):281–291. doi: 10.1023/B:BREA.0000025418.88785.2b PubMedCrossRefGoogle Scholar
  10. 10.
    Martin MD et al (2008) Effect of ablation or inhibition of stromal matrix metalloproteinase-9 lung metastasis in a breast cancer model is dependent on genetic background. Cancer Res 68:6251–6259Google Scholar
  11. 11.
    Guy CT, Cardiff RD, Muller WJ (1992) Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease. Mol Cell Biol 12(3):954–961PubMedGoogle Scholar
  12. 12.
    Liu M, Skinner SJ, Xu J et al (1992) Stimulation of fetal rat lung cell proliferation in vitro by mechanical stretch. Am J Physiol 263(3 Pt 1):L376–L383PubMedGoogle Scholar
  13. 13.
    Simpson LL, Tanswell AK, Joneja MG (1985) Epithelial cell differentiation in organotypic cultures of fetal rat lung. Am J Anat 172(1):31–40. doi: 10.1002/aja.1001720103 PubMedCrossRefGoogle Scholar
  14. 14.
    Mourgeon E, Isowa N, Keshavjee S et al (2000) Mechanical stretch stimulates macrophage inflammatory protein-2 secretion from fetal rat lung cells. Am J Physiol Lung Cell Mol Physiol 279(4):L699–L706PubMedGoogle Scholar
  15. 15.
    Sasser AK, Mundy BL, Smith KM et al (2007) Human bone marrow stromal cells enhance breast cancer cell growth rates in a cell line-dependent manner when evaluated in 3D tumor environments. Cancer Lett 254(2):255–264. doi: 10.1016/j.canlet.2007.03.012 PubMedCrossRefGoogle Scholar
  16. 16.
    Bissell MJ, Radisky D (2001) Putting tumours in context. Nat Rev Cancer 1:46–54PubMedCrossRefGoogle Scholar
  17. 17.
    Becker JL, Blanchard DK (2007) Characterization of primary breast carcinomas grown in three-dimensional cultures. J Surg Res 142(2):256–262. doi: 10.1016/j.jss.2007.03.016 PubMedCrossRefGoogle Scholar
  18. 18.
    Novaro V, Roskelley CD, Bissell MJ (2003) Collagen-IV and laminin-1 regulate estrogen receptor alpha expression and function in mouse mammary epithelial cells. J Cell Sci 116(Pt 14):2975–2986. doi: 10.1242/jcs.00523 PubMedCrossRefGoogle Scholar
  19. 19.
    Lee GY, Kenny PA, Lee EH et al (2007) Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods 4(4):359–365. doi: 10.1038/nmeth1015 PubMedCrossRefGoogle Scholar
  20. 20.
    Ohmori T, Yang JL, Price JO et al (1998) Blockade of tumor cell transforming growth factor-betas enhances cell cycle progression and sensitizes human breast carcinoma cells to cytotoxic chemotherapy. Exp Cell Res 245(2):350–359. doi: 10.1006/excr.1998.4261 PubMedCrossRefGoogle Scholar
  21. 21.
    Luck AA, Evans AJ, Green AR et al (2008) The influence of basal phenotype on the metastatic pattern of breast cancer. Clin Oncol (R Coll Radiol) 20(1):40–45. doi: 10.1016/j.clon.2007.10.002 Google Scholar

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Michelle D. Martin
    • 1
  • Barbara Fingleton
    • 1
  • Conor C. Lynch
    • 1
    • 2
  • Sam Wells
    • 3
  • J. Oliver McIntyre
    • 1
  • David W. Piston
    • 3
  • Lynn M. Matrisian
    • 1
    Email author
  1. 1.Department of Cancer BiologyVanderbilt UniversityNashvilleUSA
  2. 2.Department of Orthopaedics and RehabilitationVanderbilt UniversityNashvilleUSA
  3. 3.Department of Molecular Physiology and BiophysicsVanderbilt UniversityNashvilleUSA

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