Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Studying primary tumor–associated fibroblast involvement in cancer metastasis in mice

Nature Protocols volume 7, pages 756–762 (2012)Cite this article

Subjects

Abstract

Stromal cells have been studied extensively in the primary tumor microenvironment. In addition, mesenchymal stromal cells may participate in several steps of the metastatic cascade. Studying this interaction requires methods to distinguish and target stromal cells originating from the primary tumor versus their counterparts in the metastatic site. Here we illustrate a model of human tumor stromal cell—mouse cancer cell coimplantation. This model can be used to selectively deplete human stromal cells (using diphtheria toxin, DT) without affecting mouse cancer cells or host-derived stromal cells. Establishment of novel genetic models (e.g., transgenic expression of the DT receptor in specific cells) may eventually allow analogous models using syngeneic cells. Studying the role of stromal cells in metastasis using the model outlined above may take 8 weeks.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Design of the experiment.
Figure 2: Isolation of tumor-associated fibroblasts (TAFs) from breast cancer specimens.
Figure 3: Spontaneous metastasis of the passenger stromal cells in the coimplantation model.
Figure 4: Contribution of circulating stromal cells to spontaneous tumor metastases.

Similar content being viewed by others

References

  1. Bhowmick, N.A. et al. TGF-β signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia. Science 303, 848–851 (2004).

    Article  CAS  PubMed  Google Scholar 

  2. Jacobs, T.W., Byrne, C., Colditz, G., Connolly, J.L. & Schnitt, S.J. Radial scars in benign breast-biopsy specimens and the risk of breast cancer. N. Engl. J. Med. 340, 430–436 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Elenbaas, B. et al. Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells. Genes Dev. 15, 50–65 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bhowmick, N.A., Neilson, E.G. & Moses, H.L. Stromal fibroblasts in cancer initiation and progression. Nature 432, 332–337 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Olumi, A.F. et al. Carcinoma-associated fibroblasts direct tumor progression of initiated human prostatic epithelium. Cancer Res. 59, 5002–5011 (1999).

    CAS  PubMed  Google Scholar 

  6. Orimo, A. et al. Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion. Cell 121, 335–348 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Tlsty, T.D. Stromal cells can contribute oncogenic signals. Semin. Cancer Biol. 11, 97–104 (2001).

    Article  CAS  PubMed  Google Scholar 

  8. Kalluri, R. & Zeisberg, M. Fibroblasts in cancer. Nat. Rev. Cancer 6, 392–401 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. O'Connell, J.T. et al. VEGF-A and Tenascin-C produced by S100A4+ stromal cells are important for metastatic colonization. Proc. Natl. Acad. Sci. USA 108, 16002–16007 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Podsypanina, K. et al. Seeding and propagation of untransformed mouse mammary cells in the lung. Science 321, 1841–1844 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. 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).

    Article  CAS  PubMed  Google Scholar 

  12. Kaplan, R.N. et al. VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438, 820–827 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kim, S. et al. Carcinoma-produced factors activate myeloid cells through TLR2 to stimulate metastasis. Nature 457, 102–106 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Deventer, K., Pozo, O.J., Van Eenoo, P. & Delbeke, F.T. Development and validation of an LC-MS/MS method for the quantification of ephedrines in urine. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877, 369–374 (2009).

    Article  CAS  PubMed  Google Scholar 

  15. Al-Mehdi, A.B. et al. Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis. Nat. Med. 6, 100–102 (2000).

    Article  CAS  PubMed  Google Scholar 

  16. Liotta, L.A., Saidel, M.G. & Kleinerman, J. The significance of hematogenous tumor cell clumps in the metastatic process. Cancer Res. 36, 889–894 (1976).

    CAS  PubMed  Google Scholar 

  17. Fidler, I.J. The relationship of embolic homogeneity, number, size and viability to the incidence of experimental metastasis. Eur. J. Cancer 9, 223–227 (1973).

    Article  CAS  PubMed  Google Scholar 

  18. Ruiter, D.J., van Krieken, J.H., van Muijen, G.N. & deWaal, R.M. Tumour metastasis: is tissue an issue? Lancet Oncol. 2, 109–112 (2001).

    Article  CAS  PubMed  Google Scholar 

  19. Fidler, I.J. The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited. Nat. Rev. Cancer 3, 453–458 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Sahai, E. Illuminating the metastatic process. Nat. Rev. Cancer 7, 737–749 (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Duda, D.G. et al. Malignant cells facilitate lung metastasis by bringing their own soil. Proc. Natl. Acad. Sci. USA 107, 21677–21682 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Duyverman, A.M.M.J., Kohno, M., Duda, D.G., Jain, R.K. & Fukumura, D. A transient parabiosis skin transplantation model in mice. Nat. Protoc. 7, 763–770 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Duyverman, A.M.M.J. et al. An isolated tumor perfusion model in mice. Nat. Protoc. 7, 749–755 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Arbiser, J.L. et al. Isolation of mouse stromal cells associated with a human tumor using differential diphtheria toxin sensitivity. Am. J. Pathol. 155, 723–729 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Padera, T.P. et al. Pathology: cancer cells compress intratumour vessels. Nature 427, 695 (2004).

    Article  CAS  PubMed  Google Scholar 

  26. Padera, T.P. et al. Lymphatic metastasis in the absence of functional intratumor lymphatics. Science 296, 1883–1886 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. Dawson, M.R., Duda, D.G., Fukumura, D. & Jain, R.K. VEGFR1-activity-independent metastasis formation. Nature 461, E4–E5 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hiratsuka, S. et al. C-X-C receptor type 4 promotes metastasis by activating p38 mitogen-activated protein kinase in myeloid differentiation antigen (Gr-1)-positive cells. Proc. Natl. Acad. Sci. USA 108, 302–307 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Tian, B., Han, L., Kleidon, J. & Henke, C. An HSV-TK transgenic mouse model to evaluate elimination of fibroblasts for fibrosis therapy. Am. J. Pathol. 163, 789–801 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kassen, A. et al. Stromal cells of the human prostate: initial isolation and characterization. Prostate 28, 89–97 (1996).

    Article  CAS  PubMed  Google Scholar 

  31. Mazzocca, A. et al. Tumor-secreted lysophostatidic acid accelerates hepatocellular carcinoma progression by promoting differentiation of peritumoral fibroblasts in myofibroblasts. Hepatology 54, 920–930 (2011).

    Article  CAS  PubMed  Google Scholar 

  32. Welch, D.R. Technical considerations for studying cancer metastasis in vivo. Clin. Exp. Metastasis 15, 272–306 (1997).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The work of the authors is supported by US National Cancer Institute grants P01-CA80124, R01-CA115767, R01-CA85140, R01-CA126642 and T32-CA73479 (R.K.J.), R01-CA96915 (D.F.), R21-CA139168 and R01-CA159258 (D.G.D.) and Federal Share Proton Beam Program grants (R.K.J., D.F. and D.G.D.); Department of Defense Innovator Award W81XWH-10-1-0016 (R.K.J.) and Predoctoral Fellowship W81XWH-06-1-0781 (A.M.M.J.D.); American Cancer Society grant RSG-11-073-01TBG (D.G.D.); and Stichting Michael Van Vloten Fonds and the Stichting Jo Kolk (A.M.M.J.D.). We acknowledge the outstanding technical assistance of J. Kahn, S. Roberge and P. Huang with animal models.

Author information

Author notes

  1. Annique M M J Duyverman & Ernst J A Steller

    Present address: Present address: University Medical Center Utrecht, Utrecht, The Netherlands.,

Authors and Affiliations

  1. Department of Radiation Oncology, Edwin L. Steele Laboratory for Tumor Biology, Massachusetts General Hospital (MGH) and Harvard Medical School, Boston, Massachusetts, USA

    Annique M M J Duyverman, Ernst J A Steller, Dai Fukumura, Rakesh K Jain & Dan G Duda

Authors
  1. Annique M M J Duyverman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ernst J A Steller
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Dai Fukumura
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Rakesh K Jain
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dan G Duda
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.G.D., D.F. and R.K.J. designed the studies; A.M.M.J.D. and E.J.A.S. performed the experiments; D.G.D., D.F., A.M.M.J.D. and R.K.J. analyzed the data; and A.M.M.J.D., R.K.J. and D.G.D. edited the manuscript.

Corresponding author

Correspondence to Dan G Duda.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Duyverman, A., Steller, E., Fukumura, D. et al. Studying primary tumor–associated fibroblast involvement in cancer metastasis in mice. Nat Protoc 7, 756–762 (2012). https://doi.org/10.1038/nprot.2012.031

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2012.031

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer