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KLF4-dependent perivascular cell plasticity mediates pre-metastatic niche formation and metastasis

Abstract

A deeper understanding of the metastatic process is required for the development of new therapies that improve patient survival. Metastatic tumor cell growth and survival in distant organs is facilitated by the formation of a pre-metastatic niche that is composed of hematopoietic cells, stromal cells and extracellular matrix (ECM). Perivascular cells, including vascular smooth muscle cells (vSMCs) and pericytes, are involved in new vessel formation and in promoting stem cell maintenance and proliferation. Given the well-described plasticity of perivascular cells, we hypothesized that perivascular cells similarly regulate tumor cell fate at metastatic sites. We used perivascular-cell-specific and pericyte-specific lineage-tracing models to trace the fate of perivascular cells in the pre-metastatic and metastatic microenvironments. We show that perivascular cells lose the expression of traditional vSMC and pericyte markers in response to tumor-secreted factors and exhibit increased proliferation, migration and ECM synthesis. Increased expression of the pluripotency gene Klf4 in these phenotypically switched perivascular cells promoted a less differentiated state, characterized by enhanced ECM production, that established a pro-metastatic fibronectin-rich environment. Genetic inactivation of Klf4 in perivascular cells decreased formation of a pre-metastatic niche and metastasis. Our data revealed a previously unidentified role for perivascular cells in pre-metastatic niche formation and uncovered novel strategies for limiting metastasis.

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Figure 1: Perivascular phenotypic switching is activated in the pre-metastatic lung.
Figure 2: NG2-expressing pericytes are activated in the pre-metastatic lung.
Figure 3: Metastatic-tumor-derived factors promote activation of KLF4-dependent perivascular cell phenotypic switching.
Figure 4: Activated smooth muscle cells secrete KLF4-dependent fibronectin-containing ECM that promotes tumor adhesion, migration and proliferation.
Figure 5: Perivascular-cell-specific Klf4 deletion decreases metastasis.
Figure 6: Inhibition of tumor cell binding to fibronectin recapitulates perivascular KLF4-dependent metastasis.

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References

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Giles, A.J. et al. Activation of hematopoietic stem–progenitor cells promotes immunosuppression within the pre-metastatic niche. Cancer Res. 76, 1335–1347 (2016).

    Article  CAS  PubMed  Google Scholar 

  3. Smith, H.A. & Kang, Y. The metastasis-promoting roles of tumor-associated immune cells. J. Mol. Med. (Berl.) 91, 411–429 (2013).

    Article  CAS  Google Scholar 

  4. Sceneay, J., Parker, B.S., Smyth, M.J. & Möller, A. Hypoxia-driven immunosuppression contributes to the pre-metastatic niche. OncoImmunology 2, e22355 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  5. McAllister, S.S. & Weinberg, R.A. The tumor-induced systemic environment as a critical regulator of cancer progression and metastasis. Nat. Cell Biol. 16, 717–727 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ehling, M. & Mazzone, M. Vessel normalization in the spot-LIGHT of cancer treatment. Trends Mol. Med. 22, 85–87 (2016).

    Article  PubMed  Google Scholar 

  7. Gomez, D. & Owens, G.K. Smooth muscle cell phenotypic switching in atherosclerosis. Cardiovasc. Res. 95, 156–164 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Alexander, M.R. & Owens, G.K. Epigenetic control of smooth muscle cell differentiation and phenotypic switching in vascular development and disease. Annu. Rev. Physiol. 74, 13–40 (2012).

    Article  CAS  PubMed  Google Scholar 

  9. Armulik, A., Abramsson, A. & Betsholtz, C. Endothelial–pericyte interactions. Circ. Res. 97, 512–523 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Khan, J.A. et al. Fetal liver hematopoietic stem cell niches associate with portal vessels. Science 351, 176–180 (2016).

    Article  CAS  PubMed  Google Scholar 

  11. Kunisaki, Y. et al. Arteriolar niches maintain hematopoietic stem cell quiescence. Nature 502, 637–643 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Goel, S., Wong, A.H.-K. & Jain, R.K. Vascular normalization as a therapeutic strategy for malignant and nonmalignant disease. Cold Spring Harb. Perspect. Med. 2, a006486 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Hamzah, J. et al. Vascular normalization in Rgs5-deficient tumors promotes immune destruction. Nature 453, 410–414 (2008).

    Article  CAS  PubMed  Google Scholar 

  14. Bennett, M.R., Sinha, S. & Owens, G.K. Vascular smooth muscle cells in atherosclerosis. Circ. Res. 118, 692–702 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Armulik, A., Genové, G. & Betsholtz, C. Pericytes: developmental, physiological and pathological perspectives, problems and promises. Dev. Cell 21, 193–215 (2011).

    Article  CAS  PubMed  Google Scholar 

  16. Salmon, M. et al. KLF4 regulates abdominal aortic aneurysm morphology, and deletion attenuates aneurysm formation. Circulation 128 (Suppl. 1), S163–S174 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Shankman, L.S. et al. KLF4-dependent phenotypic modulation of smooth muscle cells has a key role in atherosclerotic plaque pathogenesis. Nat. Med. 21, 628–637 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Cherepanova, O.A. et al. Activation of the pluripotency factor OCT4 in smooth muscle cells is atheroprotective. Nat. Med. 22, 657–665 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gomez, D., Shankman, L.S., Nguyen, A.T. & Owens, G.K. Detection of histone modifications at specific gene loci in single cells in histological sections. Nat. Methods 10, 171–177 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kim, K.H. & Sederstrom, J.M. Assaying cell cycle status using flow cytometry. Curr. Protoc. Mol. Biol. 111, 28.6.1–28.6.11 (2015).

    Article  Google Scholar 

  21. Rock, J.R. et al. Multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition. Proc. Natl. Acad. Sci. USA 108, E1475–E1483 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Salmon, M., Gomez, D., Greene, E., Shankman, L. & Owens, G.K. Cooperative binding of KLF4, pELK-1 and HDAC2 to a G/C repressor element in the SM22-α promoter mediates transcriptional silencing during SMC phenotypic switching in vivo. Circ. Res. 111, 685–696 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Yoshida, T., Gan, Q. & Owens, G.K. Kruppel-like factor 4, Elk-1 and histone deacetylases cooperatively suppress smooth muscle cell differentiation markers in response to oxidized phospholipids. Am. J. Physiol. Cell Physiol. 295, C1175–C1182 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hoshino, A. et al. Tumor exosome integrins determine organotropic metastasis. Nature 527, 329–335 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gajos-Michniewicz, A., Duechler, M. & Czyz, M. miRNA in melanoma-derived exosomes. Cancer Lett. 347, 29–37 (2014).

    Article  CAS  PubMed  Google Scholar 

  26. Rafii, S. & Lyden, D. S100 chemokines mediate bookmarking of pre-metastatic niches. Nat. Cell Biol. 8, 1321–1323 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Deaton, R.A., Gan, Q. & Owens, G.K. Sp1-dependent activation of KLF4 is required for PDGF-BB-induced phenotypic modulation of smooth muscle. Am. J. Physiol. Heart Circ. Physiol. 296, H1027–H1037 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Yang, M., Du, Y., Xu, Z. & Jiang, Y. Functional effects of WNT1-inducible signaling pathway protein 1 on bronchial smooth muscle cell migration and proliferation in OVA-induced airway remodeling. Inflammation 39, 16–29 (2016).

    Article  CAS  PubMed  Google Scholar 

  29. Reddy, V.S., Valente, A.J., Delafontaine, P. & Chandrasekar, B. Interleukin-18–WNT1-inducible signaling pathway protein 1 signaling mediates human saphenous vein smooth muscle cell proliferation. J. Cell. Physiol. 226, 3303–3315 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Le, C.T.K. et al. Synergistic actions of blocking angiopoietin-2 and tumor necrosis factor–α in suppressing remodeling of blood vessels and lymphatics in airway inflammation. Am. J. Pathol. 185, 2949–2968 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Keskin, D. et al. Targeting vascular pericytes in hypoxic tumors increases lung metastasis via angiopoietin-2. Cell Rep. 10, 1066–1081 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Alexander, M.R., Murgai, M., Moehle, C.W. & Owens, G.K. Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms. Physiol. Genomics 44, 417–429 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Alexander, M.R. et al. Genetic inactivation of IL-1 signaling enhances atherosclerotic plaque instability and reduces outward vessel remodeling in advanced atherosclerosis in mice. J. Clin. Invest. 122, 70–79 (2012).

    Article  CAS  PubMed  Google Scholar 

  34. Dulauroy, S., Di Carlo, S.E., Langa, F., Eberl, G. & Peduto, L. Lineage tracing and genetic ablation of ADAM12+ perivascular cells identify a major source of profibrotic cells during acute tissue injury. Nat. Med. 18, 1262–1270 (2012).

    Article  CAS  PubMed  Google Scholar 

  35. Laklai, H. et al. Genotype tunes pancreatic ductal adenocarcinoma tissue tension to induce matricellular fibrosis and tumor progression. Nat. Med. 22, 497–505 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bissell, M.J. & Radisky, D. Putting tumors in context. Nat. Rev. Cancer 1, 46–54 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Kessenbrock, K., Plaks, V. & Werb, Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141, 52–67 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Cox, T.R. et al. LOX-mediated collagen cross-linking is responsible for fibrosis-enhanced metastasis. Cancer Res. 73, 1721–1732 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Del Pozo Martin, Y. et al. Mesenchymal cancer cell–stroma cross-talk promotes niche activation, epithelial reversion and metastatic colonization. Cell Rep. 13, 2456–2469 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Oskarsson, T., Batlle, E. & Massagué, J. Metastatic stem cells: sources, niches and vital pathways. Cell Stem Cell 14, 306–321 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. LeBleu, V.S. et al. Origin and function of myofibroblasts in kidney fibrosis. Nat. Med. 19, 1047–1053 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Baluk, P., Falcón, B.L., Hashizume, H., Sennino, B. & McDonald, D.M. in Tumor Angiogenesis 557–576 (Springer, 2008).

  43. Hanahan, D. & Folkman, J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell 86, 353–364 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Bergers, G., Song, S., Meyer-Morse, N., Bergsland, E. & Hanahan, D. Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J. Clin. Invest. 111, 1287–1295 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Morikawa, S. et al. Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors. Am. J. Pathol. 160, 985–1000 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  46. Fukumura, D. & Jain, R.K. Tumor microvasculature and microenvironment: targets for anti-angiogenesis and normalization. Microvasc. Res. 74, 72–84 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Chambers, A.F., Groom, A.C. & MacDonald, I.C. Dissemination and growth of cancer cells in metastatic sites. Nat. Rev. Cancer 2, 563–572 (2002).

    Article  CAS  PubMed  Google Scholar 

  48. Özdemir, B.C. et al. Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell 25, 719–734 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Murgai, M., Giles, A. & Kaplan, R. Physiological, tumor and metastatic niches: opportunities and challenges for targeting the tumor microenvironment. Crit. Rev. Oncog. 20, 301–314 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  50. Hanoun, M. et al. Acute myelogenous leukemia–induced sympathetic neuropathy promotes malignancy in an altered hematopoietic stem cell niche. Cell Stem Cell 15, 365–375 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Butler, J.M., Kobayashi, H. & Rafii, S. Instructive role of the vascular niche in promoting tumor growth and tissue repair by angiocrine factors. Nat. Rev. Cancer 10, 138–146 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Kusumbe, A.P. et al. Age-dependent modulation of vascular niches for hematopoietic stem cells. Nature 532, 380–384 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Calabrese, C. et al. A perivascular niche for brain tumor stem cells. Cancer Cell 11, 69–82 (2007).

    Article  CAS  PubMed  Google Scholar 

  54. Wculek, S.K. & Malanchi, I. Neutrophils support lung colonization of metastasis-initiating breast cancer cells. Nature 528, 413–417 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Yu, F. et al. Kruppel-like factor 4 (KLF4) is required for maintenance of breast cancer stem cells and for cell migration and invasion. Oncogene 30, 2161–2172 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Wei, D., Kanai, M., Huang, S. & Xie, K. Emerging role of KLF4 in human gastrointestinal cancer. Carcinogenesis 27, 23–31 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Pandya, A.Y. et al. Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer. Clin. Cancer Res. 10, 2709–2719 (2004).

    Article  CAS  PubMed  Google Scholar 

  58. Meadors, J.L. et al. Murine rhabdomyosarcoma is immunogenic and responsive to T cell–based immunotherapy. Pediatr. Blood Cancer 57, 921–929 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Carpenter, A.E. et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 7, R100 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Faul, F., Erdfelder, E., Buchner, A. & Lang, A.-G. Statistical power analyses using G*Power 3.1: tests for correlation and regression analyses. Behav. Res. Methods 41, 1149–1160 (2009).

    Article  PubMed  Google Scholar 

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Acknowledgements

We thank K. McKinnon and C. Thiele for their advice, and Z. Liu, M. Kasai, J. Zhu and K. Bhatt for their technical assistance. This work was supported by the US National Institutes of Health (grants 1ZIABC011332-07, 1ZIABC011334-07, NIH HL057353 and NIH HL135018).

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M.M. conducted most of the experiments, performed data analysis, generated most of the experimental mice and was the primary writer of the manuscript; W.J. conducted in vivo metastasis studies and in vitro matrix experiments, generated RNA for sequencing and analyzed exosomal content by western blot; M.E. conducted in vitro immunostaining and western blot experiments; J.K. assisted with the generation of experimental mice and conducted image analysis; D.W.B. and S.K. assisted with mouse experiments, and generated and analyzed flow cytometry data; M.M.M. analyzed pathological tissue specimens; M.K. assisted with confocal image acquisition, analysis and figure preparation; H.L. and J.F.S. analyzed RNA-seq data and assisted with figure preparation; O.A.C. and G.K.O. generated preliminary data, provided initial experimental mice and provided advice throughout; R.N.K. supervised the project and provided guidance on experimental design, data interpretation and writing of the manuscript.

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Correspondence to Rosandra N Kaplan.

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Murgai, M., Ju, W., Eason, M. et al. KLF4-dependent perivascular cell plasticity mediates pre-metastatic niche formation and metastasis. Nat Med 23, 1176–1190 (2017). https://doi.org/10.1038/nm.4400

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