Skip to main content
SpringerLink
Log in

Analysis of Epithelial–Mesenchymal Transition Induced by Transforming Growth Factor β

  • Protocol
TGF-β Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1344))

Abstract

In recent years, the importance of the cell biological process of epithelial–mesenchymal transition (EMT) has been established via an exponentially growing number of reports. EMT has been documented during embryonic development, tissue fibrosis, and cancer progression in vitro, in animal models in vivo and in human specimens. EMT relates to many molecular and cellular alterations that occur when epithelial cells undergo a switch in differentiation that generates mesenchymal-like cells with newly acquired migratory and invasive properties. In addition, EMT relates to a nuclear reprogramming similar to the one occurring in the generation of induced pluripotent stem cells. Via such a process, EMT is gradually established to promote the generation and maintenance of adult tissue stem cells which under disease states such as cancer, are known as cancer stem cells. EMT is induced by developmental growth factors, oncogenes, radiation, and hypoxia. A prominent growth factor that causes EMT is transforming growth factor β (TGF-β).

A series of molecular and cellular techniques can be applied to define and characterize the state of EMT in diverse biological samples. These methods range from DNA and RNA-based techniques that measure the expression of key EMT regulators and markers of epithelial or mesenchymal differentiation to functional assays of cell mobility, invasiveness and in vitro stemness. This chapter focuses on EMT induced by TGF-β and provides authoritative protocols and relevant reagents and citations of key publications aiming at assisting newcomers that enter this prolific area of biomedical sciences, and offering a useful reference tool to pioneers and aficionados of the field.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Hay ED (1995) An overview of epithelio-mesenchymal transformation. Acta Anat 154(1):8–20

    Article  CAS  PubMed  Google Scholar 

  2. Thiery JP, Acloque H, Huang RY, Nieto MA (2009) Epithelial-mesenchymal transitions in development and disease. Cell 139(5):871–890

    Article  CAS  PubMed  Google Scholar 

  3. Savagner P, Kusewitt DF, Carver EA, Magnino F, Choi C, Gridley T et al (2005) Developmental transcription factor slug is required for effective re-epithelialization by adult keratinocytes. J Cell Physiol 202(3):858–866

    Article  CAS  PubMed  Google Scholar 

  4. Iwano M, Plieth D, Danoff TM, Xue C, Okada H, Neilson EG (2002) Evidence that fibroblasts derive from epithelium during tissue fibrosis. J Clin Invest 110(3):341–350, PMCID: 151091

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  5. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY et al (2008) The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133(4):704–715, PMCID: 2728032

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  6. Morel AP, Lievre M, Thomas C, Hinkal G, Ansieau S, Puisieux A (2008) Generation of breast cancer stem cells through epithelial-mesenchymal transition. PLoS One 3(8), e2888, PMCID: 2492808

    Article  PubMed Central  PubMed  Google Scholar 

  7. Ansieau S, Bastid J, Doreau A, Morel AP, Bouchet BP, Thomas C et al (2008) Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence. Cancer Cell 14(1):79–89

    Article  CAS  PubMed  Google Scholar 

  8. Vincent T, Neve EP, Johnson JR, Kukalev A, Rojo F, Albanell J et al (2009) A SNAIL1-SMAD3/4 transcriptional repressor complex promotes TGF-β mediated epithelial-mesenchymal transition. Nat Cell Biol 11(8):943–950

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Miettinen PJ, Ebner R, Lopez AR, Derynck R (1994) TGF-β induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors. J Cell Biol 127(6 Pt 2):2021–2036, PMCID: 2120317

    Article  CAS  PubMed  Google Scholar 

  10. Piek E, Moustakas A, Kurisaki A, Heldin C-H, ten Dijke P (1999) TGF-β type I receptor/ALK-5 and Smad proteins mediate epithelial to mesenchymal transdifferentiation in NMuMG breast epithelial cells. J Cell Sci 112(Pt 24):4557–4568

    CAS  PubMed  Google Scholar 

  11. Valcourt U, Kowanetz M, Niimi H, Heldin C-H, Moustakas A (2005) TGF-β and the Smad signaling pathway support transcriptomic reprogramming during epithelial-mesenchymal cell transition. Mol Biol Cell 16(4):1987–2002, PMCID: 1073677

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Mu Y, Sundar R, Thakur N, Ekman M, Gudey SK, Yakymovych M et al (2011) TRAF6 ubiquitinates TGFβ type I receptor to promote its cleavage and nuclear translocation in cancer. Nat Commun 2:330, PMCID: 3113296

    Article  PubMed Central  PubMed  Google Scholar 

  13. Bakin AV, Tomlinson AK, Bhowmick NA, Moses HL, Arteaga CL (2000) Phosphatidylinositol 3-kinase function is required for transforming growth factor β-mediated epithelial to mesenchymal transition and cell migration. J Biol Chem 275(47):36803–36810

    Article  CAS  PubMed  Google Scholar 

  14. Bhowmick NA, Ghiassi M, Bakin A, Aakre M, Lundquist CA, Engel ME et al (2001) Transforming growth factor-β1 mediates epithelial to mesenchymal transdifferentiation through a RhoA-dependent mechanism. Mol Biol Cell 12(1):27–36, PMCID: 30565

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. Bhowmick NA, Zent R, Ghiassi M, McDonnell M, Moses HL (2001) Integrin β 1 signaling is necessary for transforming growth factor-β activation of p38MAPK and epithelial plasticity. J Biol Chem 276(50):46707–46713

    Article  CAS  PubMed  Google Scholar 

  16. Cicchini C, Laudadio I, Citarella F, Corazzari M, Steindler C, Conigliaro A et al (2008) TGFβ-induced EMT requires focal adhesion kinase (FAK) signaling. Exp Cell Res 314(1):143–152

    Article  CAS  PubMed  Google Scholar 

  17. Bakin AV, Rinehart C, Tomlinson AK, Arteaga CL (2002) p38 mitogen-activated protein kinase is required for TGFβ-mediated fibroblastic transdifferentiation and cell migration. J Cell Sci 115(Pt 15):3193–3206

    CAS  PubMed  Google Scholar 

  18. Galliher AJ, Schiemann WP (2007) Src phosphorylates Tyr284 in TGF-β type II receptor and regulates TGF-β stimulation of p38 MAPK during breast cancer cell proliferation and invasion. Cancer Res 67(8):3752–3758

    Article  CAS  PubMed  Google Scholar 

  19. Wendt MK, Tian M, Schiemann WP (2012) Deconstructing the mechanisms and consequences of TGF-β-induced EMT during cancer progression. Cell Tissue Res 347(1):85–101

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Ozdamar B, Bose R, Barrios-Rodiles M, Wang HR, Zhang Y, Wrana JL (2005) Regulation of the polarity protein Par6 by TGFβ receptors controls epithelial cell plasticity. Science 307(5715):1603–1609

    Article  CAS  PubMed  Google Scholar 

  21. Batlle E, Sancho E, Franci C, Dominguez D, Monfar M, Baulida J et al (2000) The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nat Cell Biol 2(2):84–89

    Article  CAS  PubMed  Google Scholar 

  22. Cano A, Perez-Moreno MA, Rodrigo I, Locascio A, Blanco MJ, del Barrio MG et al (2000) The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nat Cell Biol 2(2):76–83

    Article  CAS  PubMed  Google Scholar 

  23. Hajra KM, Chen DY, Fearon ER (2002) The SLUG zinc-finger protein represses E-cadherin in breast cancer. Cancer Res 62(6):1613–1618

    CAS  PubMed  Google Scholar 

  24. Bolos V, Peinado H, Perez-Moreno MA, Fraga MF, Esteller M, Cano A (2003) The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors. J Cell Sci 116(Pt 3):499–511

    Article  CAS  PubMed  Google Scholar 

  25. Eger A, Aigner K, Sonderegger S, Dampier B, Oehler S, Schreiber M et al (2005) ΔEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells. Oncogene 24(14):2375–2385

    Article  CAS  PubMed  Google Scholar 

  26. Comijn J, Berx G, Vermassen P, Verschueren K, van Grunsven L, Bruyneel E et al (2001) The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. Mol Cell 7(6):1267–1278

    Article  CAS  PubMed  Google Scholar 

  27. Perez-Moreno MA, Locascio A, Rodrigo I, Dhondt G, Portillo F, Nieto MA et al (2001) A new role for E12/E47 in the repression of E-cadherin expression and epithelial-mesenchymal transitions. J Biol Chem 276(29):27424–27431

    Article  CAS  PubMed  Google Scholar 

  28. Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C et al (2004) Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 117(7):927–939

    Article  CAS  PubMed  Google Scholar 

  29. Kowanetz M, Valcourt U, Bergstrom R, Heldin CH, Moustakas A (2004) Id2 and Id3 define the potency of cell proliferation and differentiation responses to transforming growth factor β and bone morphogenetic protein. Mol Cell Biol 24(10):4241–4254, PMCID: 400464

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Kondo M, Cubillo E, Tobiume K, Shirakihara T, Fukuda N, Suzuki H et al (2004) A role for Id in the regulation of TGF-β-induced epithelial-mesenchymal transdifferentiation. Cell Death Differ 11(10):1092–1101

    Article  CAS  PubMed  Google Scholar 

  31. Thuault S, Valcourt U, Petersen M, Manfioletti G, Heldin C-H, Moustakas A (2006) Transforming growth factor-β employs HMGA2 to elicit epithelial-mesenchymal transition. J Cell Biol 174(2):175–183, PMCID: 2064178

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Thuault S, Tan E-J, Peinado H, Cano A, Heldin C-H, Moustakas A (2008) HMGA2 and Smads co-regulate SNAIL1 expression during induction of epithelial-to-mesenchymal transition. J Biol Chem 283(48):33437–33446, PMCID: 2662269

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Tan E-J, Thuault S, Caja L, Carletti T, Heldin C-H, Moustakas A (2012) Regulation of transcription factor Twist expression by the DNA architectural protein high mobility group A2 during epithelial-to-mesenchymal transition. J Biol Chem 287(10):7134–7145, PMCID: 3293571

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Moustakas A, Heldin C-H (2012) Induction of epithelial-mesenchymal transition by transforming growth factor β. Semin Cancer Biol 22(5–6):446–454

    Article  CAS  PubMed  Google Scholar 

  35. Massagué J (2012) TGFβ signalling in context. Nat Rev Mol Cell Biol 13(10):616–630

    Article  PubMed Central  PubMed  Google Scholar 

  36. Nieto MA (2011) The ins and outs of the epithelial to mesenchymal transition in health and disease. Annu Rev Cell Dev Biol 27:347–376

    Article  CAS  PubMed  Google Scholar 

  37. Sundqvist A, Zieba A, Vasilaki E, Herrera Hidalgo C, Söderberg O, Koinuma D et al (2013) Specific interactions between Smad proteins and AP-1 components determine TGFβ-induced breast cancer cell invasion. Oncogene 32(31):3606–3615

    Article  CAS  PubMed  Google Scholar 

  38. Brown KA, Aakre ME, Gorska AE, Price JO, Eltom SE, Pietenpol JA et al (2004) Induction by transforming growth factor-β1 of epithelial to mesenchymal transition is a rare event in vitro. Breast Cancer Res 6(3):R215–R231, PMCID: 400675

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Masuda A, Kondo M, Saito T, Yatabe Y, Kobayashi T, Okamoto M et al (1997) Establishment of human peripheral lung epithelial cell lines (HPL1) retaining differentiated characteristics and responsiveness to epidermal growth factor, hepatocyte growth factor, and transforming growth factor β1. Cancer Res 57(21):4898–4904

    CAS  PubMed  Google Scholar 

  40. Cui W, Fowlis DJ, Bryson S, Duffie E, Ireland H, Balmain A et al (1996) TGFβ1 inhibits the formation of benign skin tumors, but enhances progression to invasive spindle carcinomas in transgenic mice. Cell 86(4):531–542

    Article  CAS  PubMed  Google Scholar 

  41. Portella G, Cumming SA, Liddell J, Cui W, Ireland H, Akhurst RJ et al (1998) Transforming growth factor β is essential for spindle cell conversion of mouse skin carcinoma in vivo: implications for tumor invasion. Cell Growth Differ 9(5):393–404

    CAS  PubMed  Google Scholar 

  42. Oft M, Heider KH, Beug H (1998) TGFβ signaling is necessary for carcinoma cell invasiveness and metastasis. Curr Biol 8(23):1243–1252

    Article  CAS  PubMed  Google Scholar 

  43. Peinado H, Quintanilla M, Cano A (2003) Transforming growth factor β-1 induces snail transcription factor in epithelial cell lines: mechanisms for epithelial mesenchymal transitions. J Biol Chem 278(23):21113–21123

    Article  CAS  PubMed  Google Scholar 

  44. Huber MA, Kraut N, Beug H (2005) Molecular requirements for epithelial-mesenchymal transition during tumor progression. Curr Opin Cell Biol 17(5):548–558

    Article  CAS  PubMed  Google Scholar 

  45. Hesling C, Fattet L, Teyre G, Jury D, Gonzalo P, Lopez J et al (2011) Antagonistic regulation of EMT by TIF1γ and Smad4 in mammary epithelial cells. EMBO Rep 12(7):665–672, PMCID: 3128966

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  46. Scheel C, Eaton EN, Li SH, Chaffer CL, Reinhardt F, Kah KJ et al (2011) Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast. Cell 145(6):926–940

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  47. Vincent DF, Yan KP, Treilleux I, Gay F, Arfi V, Kaniewski B et al (2009) Inactivation of TIF1γ cooperates with Kras to induce cystic tumors of the pancreas. PLoS Genet 5(7), e1000575, PMCID: 2706992

    Article  PubMed Central  PubMed  Google Scholar 

  48. Vincent DF, Gout J, Chuvin N, Arfi V, Pommier RM, Bertolino P et al (2012) Tif1γ suppresses murine pancreatic tumoral transformation by a Smad4-independent pathway. Am J Pathol 180(6):2214–2221, PMCID: 3378851

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  49. Xie L, Law BK, Aakre ME, Edgerton M, Shyr Y, Bhowmick NA et al (2003) Transforming growth factor β-regulated gene expression in a mouse mammary gland epithelial cell line. Breast Cancer Res 5(6):R187–R198, PMCID: 314403

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  50. Jechlinger M, Grunert S, Tamir IH, Janda E, Ludemann S, Waerner T et al (2003) Expression profiling of epithelial plasticity in tumor progression. Oncogene 22(46):7155–7169

    Article  CAS  PubMed  Google Scholar 

  51. Levy L, Hill CS (2005) Smad4 dependency defines two classes of transforming growth factor β (TGF-β) target genes and distinguishes TGF-β-induced epithelial-mesenchymal transition from its antiproliferative and migratory responses. Mol Cell Biol 25(18):8108–8125, PMCID: 1234333

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  52. Gervasi M, Bianchi-Smiraglia A, Cummings M, Zheng Q, Wang D, Liu S et al (2012) JunB contributes to Id2 repression and the epithelial-mesenchymal transition in response to transforming growth factor-β. J Cell Biol 196(5):589–603, PMCID: 3307698

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  53. Moreno-Bueno G, Cubillo E, Sarrio D, Peinado H, Rodriguez-Pinilla SM, Villa S et al (2006) Genetic profiling of epithelial cells expressing E-cadherin repressors reveals a distinct role for Snail, Slug, and E47 factors in epithelial-mesenchymal transition. Cancer Res 66(19):9543–9556

    Article  CAS  PubMed  Google Scholar 

  54. Mori M, Nakagami H, Koibuchi N, Miura K, Takami Y, Koriyama H et al (2009) Zyxin mediates actin fiber reorganization in epithelial-mesenchymal transition and contributes to endocardial morphogenesis. Mol Biol Cell 20(13):3115–3124, PMCID: 2704162

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  55. Haynes J, Srivastava J, Madson N, Wittmann T, Barber DL (2011) Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression. Mol Biol Cell 22(24):4750–4764, PMCID: 3237619

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  56. Koinuma D, Tsutsumi S, Kamimura N, Imamura T, Aburatani H, Miyazono K (2009) Promoter-wide analysis of Smad4 binding sites in human epithelial cells. Cancer Sci 100(11):2133–2142

    Article  CAS  PubMed  Google Scholar 

  57. Shapiro IM, Cheng AW, Flytzanis NC, Balsamo M, Condeelis JS, Oktay MH et al (2011) An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype. PLoS Genet 7(8), e1002218, PMCID: 3158048

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  58. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G et al (2008) The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 10(5):593–601

    Article  CAS  PubMed  Google Scholar 

  59. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ et al (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17(10):1253–1270, PMCID: 196056

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  60. Gal A, Sjöblom T, Fedorova L, Imreh S, Beug H, Moustakas A (2008) Sustained TGF β exposure suppresses Smad and non-Smad signalling in mammary epithelial cells, leading to EMT and inhibition of growth arrest and apoptosis. Oncogene 27(9):1218–1230

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We acknowledge funding by the Ludwig Institute for Cancer Research, the Swedish Cancer Society and the Swedish Research Council (A.M.). We also acknowledge funding by the “Ligue Nationale Contre le Cancer” (LNCC) (L.A.), the “Comité du Rhône de la Ligue Nationale Contre le Cancer” (U.V.) and the “Institut National du Cancer” (INCa) (L.B.).

We thank all past and present members of our groups for their contributions to the scientific work emanating from our laboratories. This chapter summarizes work from a selected number of published papers, however, due to space limitations, we have been unable to include all relevant publications in our discussion. We apologize to those authors whose relevant work has not been included in this review chapter.

We declare no conflict of interest.

Author information

Authors and Affiliations

  1. Inserm U1052, Centre de Recherche en Cancérologie de Lyon, 69000, Lyon, France

    Ulrich Valcourt, Lindsay Alcaraz & Laurent Bartholin

  2. CNRS UMR 5286, Centre de Recherche en Cancérologie de Lyon, 69000, Lyon, France

    Ulrich Valcourt, Lindsay Alcaraz & Laurent Bartholin

  3. Université de Lyon, 69000, Lyon, France

    Ulrich Valcourt, Lindsay Alcaraz & Laurent Bartholin

  4. Université Lyon 1, 69000, Lyon, France

    Ulrich Valcourt, Lindsay Alcaraz & Laurent Bartholin

  5. Centre Léon Bérard, 69000, Lyon, France

    Ulrich Valcourt, Lindsay Alcaraz & Laurent Bartholin

  6. Ludwig Institute for Cancer Research, Science for Life Laboratory, Uppsala University, Box 595, 751 24, Uppsala, Sweden

    Jonathon Carthy, Yukari Okita & Aristidis Moustakas Ph.D.

  7. Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan

    Yukari Okita & Mitsuyasu Kato

  8. INSERM UMR 911 CRO2, Faculty of Pharmacy, Marseille, France

    Sylvie Thuault

  9. Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, Box 582, 751 23, Uppsala, Sweden

    Aristidis Moustakas Ph.D.

Authors
  1. Ulrich Valcourt
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jonathon Carthy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yukari Okita
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lindsay Alcaraz
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mitsuyasu Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sylvie Thuault
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Laurent Bartholin
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Aristidis Moustakas Ph.D.
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Aristidis Moustakas Ph.D. .

Editor information

Editors and Affiliations

  1. Departments of Surgery and Molecular & Cellular Biology, Baylor College of Medicine, Houston, Texas, USA

    Xin-Hua Feng

  2. Life Sciences Institute, Zhejiang University, Hangzhou, Zhejiang Proviince, China

    Pinglong Xu

  3. Department of Surgery, Baylor College of Medicine, Houston, Texas, USA

    Xia Lin

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Valcourt, U. et al. (2016). Analysis of Epithelial–Mesenchymal Transition Induced by Transforming Growth Factor β. In: Feng, XH., Xu, P., Lin, X. (eds) TGF-β Signaling. Methods in Molecular Biology, vol 1344. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2966-5_9

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2966-5_9

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2965-8

  • Online ISBN: 978-1-4939-2966-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation