Microscopic Methods for Analysis of Macrophage-Induced Tunneling Nanotubes

  • Kiersten P. Carter
  • Jeffrey E. Segall
  • Dianne CoxEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2108)


Macrophages are known to play multiple roles in the breast cancer microenvironment including the promotion of tumor cell invasion that is dependent on soluble factors or through direct contact. Macrophages can also enhance the production of Tunneling Nanotubes (TNTs) in tumor cells which can be mimicked using macrophage-conditioned medium. TNTs are long thin F-actin structures that connect two or more cells together that have been found in many different cell types including macrophages and tumor cells and have been implicated in enhancing tumor cells functions, such as invasion. Here we describe basic procedures used to stimulate tumor cell TNT formation through macrophage-conditioned medium along with methods for quantifying TNTs.

Key words

Tunneling nanotubes Macrophages Tumor cell Microscopy Conditioned medium 


  1. 1.
    Sharma VP, Beaty BT, Cox D et al (2014) An in vitro one-dimensional assay to study growth factor-regulated tumor cell-macrophage interaction. Methods Mol Biol 1172:115–123CrossRefGoogle Scholar
  2. 2.
    Yang M, McKay D, Pollard JW et al (2018) Diverse functions of macrophages in different tumor microenvironments. Cancer Res 78:5492–5503CrossRefGoogle Scholar
  3. 3.
    Davies LC, Taylor PR (2015) Tissue-resident macrophages: then and now. Immunology 144:541–548CrossRefGoogle Scholar
  4. 4.
    Wyckoff J, Wang W, Lin EY et al (2004) A paracrine loop between tumor cells and macrophages is required for tumor cell migration in mammary tumors. Cancer Res 64:7022–7029CrossRefGoogle Scholar
  5. 5.
    Dang W, Qin Z, Fan S et al (2016) miR-1207-5p suppresses lung cancer growth and metastasis by targeting CSF1. Oncotarget 7:32421–32432PubMedPubMedCentralGoogle Scholar
  6. 6.
    Lou E, Gholami S, Romin Y et al (2017) Imaging tunneling membrane tubes elucidates cell communication in tumors. Trends Cancer 3:678–685CrossRefGoogle Scholar
  7. 7.
    Hanna SJ, McCoy-Simandle K, Leung E et al (2019) Tunneling nanotubes, a novel mode of tumor cell-macrophage communication in tumor cell invasion. J Cell Sci 132(3). pii: jcs223321. Scholar
  8. 8.
    Patheja P, Sahu K (2017) Macrophage conditioned medium induced cellular network formation in MCF-7 cells through enhanced tunneling nanotube formation and tunneling nanotube mediumted release of viable cytoplasmic fragments. Exp Cell Res 355:182–193CrossRefGoogle Scholar
  9. 9.
    Carter KP, Hanna S, Genna A et al (2019) Macrophage induced tumor cell tunneling nanotubes enhance tumor cell 3D invasion. Cancer Rep, 28 August 2019.
  10. 10.
    Eugenin EA, Gaskill PJ, Berman JW (2009) Tunneling nanotubes (TNT): a potential mechanism for intercellular HIV trafficking. Commun Integr Biol 2:243–244CrossRefGoogle Scholar
  11. 11.
    Saenz-de-Santa-Maria I, Bernardo-Castineira C, Enciso E, Garcia-Moreno I et al (2017) Control of long-distance cell-to-cell communication and autophagosome transfer in squamous cell carcinoma via tunneling nanotubes. Oncotarget 8:20939–20960PubMedPubMedCentralGoogle Scholar
  12. 12.
    Cox D, Chang P, Zhang Q et al (1997) Requirements for both Rac1 and Cdc42 in membrane ruffling and phagocytosis in leukocytes. J Exp Med 186:1487–1494CrossRefGoogle Scholar
  13. 13.
    Hanna SJ, McCoy-Simandle K, Miskolci V et al (2017) The role of Rho-GTPases and actin polymerization during macrophage tunneling nanotube biogenesis. Sci Rep 7:8547CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Kiersten P. Carter
    • 1
  • Jeffrey E. Segall
    • 1
    • 2
  • Dianne Cox
    • 1
    • 2
    • 3
    Email author
  1. 1.Department of Anatomy and Structural BiologyAlbert Einstein College of MedicineBronxUSA
  2. 2.Gruss-Lipper Biophotonics CenterAlbert Einstein College of MedicineBronxUSA
  3. 3.Department of Molecular and Developmental BiologyAlbert Einstein College of MedicineBronxUSA

Personalised recommendations