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Cellular and Molecular Life Sciences

, Volume 72, Issue 19, pp 3769–3782 | Cite as

Quantification of cell fusion events human breast cancer cells and breast epithelial cells using a Cre-LoxP-based double fluorescence reporter system

  • Marieke Mohr
  • Songül Tosun
  • Wolfgang H. Arnold
  • Frank Edenhofer
  • Kurt S. Zänker
  • Thomas DittmarEmail author
Research Article

Abstract

The biological phenomenon of cell fusion plays an important role in several physiological processes, like fertilization, placentation, or wound healing/tissue regeneration, as well as pathophysiological processes, such as cancer. Despite this fact, considerably less is still known about the factors and conditions that will induce the merging of two plasma membranes. Inflammation and proliferation has been suggested as a positive trigger for cell fusion, but it remains unclear, which of the factor(s) of the inflamed microenvironment are being involved. To clarify this we developed a reliable assay to quantify the in vitro fusion frequency of cells using a fluorescence double reporter vector (pFDR) containing a LoxP-flanked HcRed/DsRed expression cassette followed by an EGFP expression cassette. Because cell fusion has been implicated in cancer progression four human breast cancer cell lines were stably transfected with a pFDR vector and were co-cultured with the stably Cre-expressing human breast epithelial cell line. Cell fusion is associated with a Cre-mediated recombination resulting in induction of EGFP expression in hybrid cells, which can be quantified by flow cytometry. By testing a panel of different cytokines, chemokines, growth factors and other compounds, including exosomes, under normoxic and hypoxic conditions our data indicate that the proinflammatory cytokine TNF-α together with hypoxia is a strong inducer of cell fusion in human MDA-MB-435 and MDA-MB-231 breast cancer cells.

Keywords

Cell fusion Breast cancer Cre-LoxP recombination Flow cytometry 

Notes

Acknowledgments

We would like to thank Dr. Oliver Thamm (Clinic for Plastic- and Reconstructive Surgery, Handsurgery, Burn Care Center, University of Witten/Herdecke, Cologne-Merheim Medical Center, Cologne, Germany) for providing us with chronic wound fluid (CWF). This work was supported by the Fritz-Bender-Foundation, Munich, Germany.

Supplementary material

18_2015_1910_MOESM1_ESM.doc (335 kb)
Supplementary data 1 These tables summarize the tested compounds, including concentration, and conditions, used in this study for each cell line. The relative fold change was calculated as described in the Material & Methods section. Shown are the mean ± STD of at least three independent experiments (DOC 335 kb)
18_2015_1910_MOESM2_ESM.tif (634 kb)
Supplementary data 2 In this figure the relative fold changes in the amount of cell debris, also containing apoptotic bodies, of the compounds and conditions, which led to an increased number of green fluorescing cells are summarized. The amount of cell debris/apoptotic bodies was determined by gating the population located in the lower left corner of a FSC-H/SSC-H dot plot diagram. Shown are the mean ± STD of at least three independent experiments (TIFF 633 kb)

References

  1. 1.
    Dittmar T (2002) Modern technologies in immune diagnosis. Infect Immun Pharmacol 1–2:17–19Google Scholar
  2. 2.
    Dittmar T, Zänker KS (2011) Cell fusion in health and disease, vol 1. Adv Exp Med Biol. Springer, DordrechtGoogle Scholar
  3. 3.
    Dittmar T, Zänker KS (2011) Cell fusion in health and disease, vol 2. Adv Exp Med Biol. Springer, DordrechtGoogle Scholar
  4. 4.
    Perez-Vargas J, Krey T, Valansi C, Avinoam O, Haouz A, Jamin M, Raveh-Barak H, Podbilewicz B, Rey FA (2014) Structural basis of eukaryotic cell–cell fusion. Cell 157(2):407–419. doi: 10.1016/j.cell.2014.02.020 CrossRefPubMedGoogle Scholar
  5. 5.
    Huppertz B, Bartz C, Kokozidou M (2006) Trophoblast fusion: fusogenic proteins, syncytins and ADAMs, and other prerequisites for syncytial fusion. Micron 37(6):509–517CrossRefPubMedGoogle Scholar
  6. 6.
    Mi S, Lee X, Li X, Veldman GM, Finnerty H, Racie L, LaVallie E, Tang XY, Edouard P, Howes S, Keith JC Jr, McCoy JM (2000) Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis. Nature 403(6771):785–789CrossRefPubMedGoogle Scholar
  7. 7.
    Podbilewicz B (2014) Virus and cell fusion mechanisms. Annu Rev Cell Dev Biol 30:111–139. doi: 10.1146/annurev-cellbio-101512-122422 CrossRefPubMedGoogle Scholar
  8. 8.
    Cornelis G, Heidmann O, Bernard-Stoecklin S, Reynaud K, Veron G, Mulot B, Dupressoir A, Heidmann T (2012) Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora. Proc Natl Acad Sci USA 109(7):E432–441. doi: 10.1073/pnas.1115346109 PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Aguilar PS, Baylies MK, Fleissner A, Helming L, Inoue N, Podbilewicz B, Wang H, Wong M (2013) Genetic basis of cell–cell fusion mechanisms. Trends Genet 29(7):427–437. doi: 10.1016/j.tig.2013.01.011 PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Zhou X, Platt JL (2011) Molecular and cellular mechanisms of mammalian cell fusion. Adv Exp Med Biol 713:33–64. doi: 10.1007/978-94-007-0763-4_4 CrossRefPubMedGoogle Scholar
  11. 11.
    Alvarez-Dolado M, Martinez-Losa M (2011) Cell fusion and tissue regeneration. Adv Exp Med Biol 713:161–175. doi: 10.1007/978-94-007-0763-4_10 CrossRefPubMedGoogle Scholar
  12. 12.
    Kozorovitskiy Y, Gould E (2003) Stem cell fusion in the brain. Nat Cell Biol 5(11):952–954CrossRefPubMedGoogle Scholar
  13. 13.
    Vassilopoulos G, Russell DW (2003) Cell fusion: an alternative to stem cell plasticity and its therapeutic implications. Curr Opin Genet Dev 13(5):480–485CrossRefPubMedGoogle Scholar
  14. 14.
    Wang X, Willenbring H, Akkari Y, Torimaru Y, Foster M, Al-Dhalimy M, Lagasse E, Finegold M, Olson S, Grompe M (2003) Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 422(6934):897–901CrossRefPubMedGoogle Scholar
  15. 15.
    Willenbring H, Bailey AS, Foster M, Akkari Y, Dorrell C, Olson S, Finegold M, Fleming WH, Grompe M (2004) Myelomonocytic cells are sufficient for therapeutic cell fusion in liver. Nat Med 10(7):744–748CrossRefPubMedGoogle Scholar
  16. 16.
    Davies PS, Powell AE, Swain JR, Wong MH (2009) Inflammation and proliferation act together to mediate intestinal cell fusion. PLoS ONE 4(8):e6530. doi: 10.1371/journal.pone.0006530 PubMedCentralCrossRefPubMedGoogle Scholar
  17. 17.
    Johansson CB, Youssef S, Koleckar K, Holbrook C, Doyonnas R, Corbel SY, Steinman L, Rossi FM, Blau HM (2008) Extensive fusion of haematopoietic cells with Purkinje neurons in response to chronic inflammation. Nat Cell Biol 10(5):575–583PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Balkwill F, Mantovani A (2001) Inflammation and cancer: back to Virchow? Lancet 357(9255):539–545. doi: 10.1016/S0140-6736(00)04046-0 CrossRefPubMedGoogle Scholar
  19. 19.
    Dvorak HF (1986) Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 315(26):1650–1659CrossRefPubMedGoogle Scholar
  20. 20.
    Dittmar T, Nagler C, Niggemann B, Zänker KS (2013) The dark side of stem cells: triggering cancer progression by cell fusion. Curr Mol Med 13(5):735–750CrossRefPubMedGoogle Scholar
  21. 21.
    Duelli D, Lazebnik Y (2003) Cell fusion: a hidden enemy? Cancer Cell 3(5):445–448CrossRefPubMedGoogle Scholar
  22. 22.
    Rachkovsky M, Sodi S, Chakraborty A, Avissar Y, Bolognia J, McNiff JM, Platt J, Bermudes D, Pawelek J (1998) Melanoma x macrophage hybrids with enhanced metastatic potential. Clin Exp Metastasis 16(4):299–312CrossRefPubMedGoogle Scholar
  23. 23.
    Lu X, Kang Y (2009) Efficient acquisition of dual metastasis organotropism to bone and lung through stable spontaneous fusion between MDA-MB-231 variants. Proc Natl Acad Sci USA 106(23):9385–9390PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Xu MH, Gao X, Luo D, Zhou XD, Xiong W, Liu GX (2014) EMT and acquisition of stem cell-like properties are involved in spontaneous formation of tumorigenic hybrids between lung cancer and bone marrow-derived mesenchymal stem cells. PLoS ONE 9(2):e87893. doi: 10.1371/journal.pone.0087893 PubMedCentralCrossRefPubMedGoogle Scholar
  25. 25.
    Miller FR, Mohamed AN, McEachern D (1989) Production of a more aggressive tumor cell variant by spontaneous fusion of two mouse tumor subpopulations. Cancer Res 49(15):4316–4321PubMedGoogle Scholar
  26. 26.
    Dittmar T, Schwitalla S, Seidel J, Haverkampf S, Reith G, Meyer-Staeckling S, Brandt BH, Niggemann B, Zanker KS (2011) Characterization of hybrid cells derived from spontaneous fusion events between breast epithelial cells exhibiting stem-like characteristics and breast cancer cells. Clin Exp Metastasis 28(1):75–90CrossRefPubMedGoogle Scholar
  27. 27.
    Carloni V, Mazzocca A, Mello T, Galli A, Capaccioli S (2013) Cell fusion promotes chemoresistance in metastatic colon carcinoma. Oncogene 32(21):2649–2660. doi: 10.1038/onc.2012.268 CrossRefPubMedGoogle Scholar
  28. 28.
    Dittmar T, Nagler C, Schwitalla S, Reith G, Niggemann B, Zanker KS (2009) Recurrence cancer stem cells—made by cell fusion? Med Hypotheses 73(4):542–547CrossRefPubMedGoogle Scholar
  29. 29.
    Chang CC, Sun W, Cruz A, Saitoh M, Tai MH, Trosko JE (2001) A human breast epithelial cell type with stem cell characteristics as target cells for carcinogenesis. Radiat Res 155(1 Pt 2):201–207CrossRefPubMedGoogle Scholar
  30. 30.
    Nolden L, Edenhofer F, Haupt S, Koch P, Wunderlich FT, Siemen H, Brustle O (2006) Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase. Nat Methods 3(6):461–467. doi: 10.1038/nmeth884 CrossRefPubMedGoogle Scholar
  31. 31.
    Somers A, Jean JC, Sommer CA, Omari A, Ford CC, Mills JA, Ying L, Sommer AG, Jean JM, Smith BW, Lafyatis R, Demierre MF, Weiss DJ, French DL, Gadue P, Murphy GJ, Mostoslavsky G, Kotton DN (2010) Generation of transgene-free lung disease-specific human induced pluripotent stem cells using a single excisable lentiviral stem cell cassette. Stem Cells 28(10):1728–1740. doi: 10.1002/stem.495 PubMedCentralCrossRefPubMedGoogle Scholar
  32. 32.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  33. 33.
    Koenen P, Spanholtz TA, Maegele M, Sturmer E, Brockamp T, Neugebauer E, Thamm OC (2015) Acute and chronic wound fluids inversely influence adipose-derived stem cell function: molecular insights into impaired wound healing. Int Wound J 12(1):10–16. doi: 10.1111/iwj.12039 CrossRefPubMedGoogle Scholar
  34. 34.
    Voss MJ, Moller MF, Powe DG, Niggemann B, Zanker KS, Entschladen F (2011) Luminal and basal-like breast cancer cells show increased migration induced by hypoxia, mediated by an autocrine mechanism. BMC Cancer 11:158. doi: 10.1186/1471-2407-11-158 PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Alvarez-Dolado M, Pardal R, Garcia-Verdugo JM, Fike JR, Lee HO, Pfeffer K, Lois C, Morrison SJ, Alvarez-Buylla A (2003) Fusion of bone-marrow-derived cells with Purkinje neurons, cardiomyocytes and hepatocytes. Nature 425(6961):968–973CrossRefPubMedGoogle Scholar
  36. 36.
    Sprangers AJ, Freeman BT, Kouris NA, Ogle BM (2012) A Cre-Lox P recombination approach for the detection of cell fusion in vivo. J Vis Exp 59:e3581. doi: 10.3791/3581 PubMedGoogle Scholar
  37. 37.
    Heyder C, Gloria-Maercker E, Hatzmann W, Zänker KS, Dittmar T (2006) Circulating cancer cells: Flow cytometry, video microscopy and confocal laser scanning microscopy. In: Hayat MA (ed) Handbook of immunohistochemistry and in situ hybridization of human carcinomas, vol 4., Molecular genetics, gastrointestinal carcinoma, and ovarian carcinoma, vol 4Elsevier Academic Press, Burlington, pp 77–88Google Scholar
  38. 38.
    Record M (2014) Intercellular communication by exosomes in placenta: a possible role in cell fusion? Placenta 35(5):297–302. doi: 10.1016/j.placenta.2014.02.009 CrossRefPubMedGoogle Scholar
  39. 39.
    Sinkovics JG (2011) Horizontal gene transfers with or without cell fusions in all categories of the living matter. Adv Exp Med Biol 714:5–89. doi: 10.1007/978-94-007-0782-5_2 CrossRefPubMedGoogle Scholar
  40. 40.
    Bjerkvig R, Tysnes BB, Aboody KS, Najbauer J, Terzis AJ (2005) Opinion: the origin of the cancer stem cell: current controversies and new insights. Nat Rev Cancer 5(11):899–904CrossRefPubMedGoogle Scholar
  41. 41.
    Yan B, Wang H, Li F, Li CY (2006) Regulation of mammalian horizontal gene transfer by apoptotic DNA fragmentation. Br J Cancer 95(12):1696–1700. doi: 10.1038/sj.bjc.6603484 PubMedCentralCrossRefPubMedGoogle Scholar
  42. 42.
    Bergsmedh A, Szeles A, Henriksson M, Bratt A, Folkman MJ, Spetz AL, Holmgren L (2001) Horizontal transfer of oncogenes by uptake of apoptotic bodies. Proc Natl Acad Sci USA 98(11):6407–6411PubMedCentralCrossRefPubMedGoogle Scholar
  43. 43.
    Bergsmedh A, Ehnfors J, Spetz AL, Holmgren L (2007) A Cre-loxP based system for studying horizontal gene transfer. FEBS Lett 581(16):2943–2946. doi: 10.1016/j.febslet.2007.05.045 CrossRefPubMedGoogle Scholar
  44. 44.
    Nagler C, Hardt C, Zänker KS, Dittmar T (2011) Co-cultivation of murine BMDCs with 67NR mouse mammary carcinoma cells give rise to highly drug resistant hybrid cells. Cancer Cell Int 11:21PubMedCentralCrossRefPubMedGoogle Scholar
  45. 45.
    Trejo-Becerril C, Perez-Cardenas E, Taja-Chayeb L, Anker P, Herrera-Goepfert R, Medina-Velazquez LA, Hidalgo-Miranda A, Perez-Montiel D, Chavez-Blanco A, Cruz-Velazquez J, Diaz-Chavez J, Gaxiola M, Duenas-Gonzalez A (2012) Cancer progression mediated by horizontal gene transfer in an in vivo model. PLoS ONE 7(12):e52754. doi: 10.1371/journal.pone.0052754 PubMedCentralCrossRefPubMedGoogle Scholar
  46. 46.
    Ehnfors J, Kost-Alimova M, Persson NL, Bergsmedh A, Castro J, Levchenko-Tegnebratt T, Yang L, Panaretakis T, Holmgren L (2009) Horizontal transfer of tumor DNA to endothelial cells in vivo. Cell Death Differ 16(5):749–757. doi: 10.1038/cdd.2009.7 CrossRefPubMedGoogle Scholar
  47. 47.
    Ozel C, Seidel J, Meyer-Staeckling S, Brandt BH, Niggemann B, Zanker KS, Dittmar T (2012) Hybrid cells derived from breast epithelial cell/breast cancer cell fusion events show a differential RAF-AKT crosstalk. Cell Commun Signal 10(1):10. doi: 10.1186/1478-811X-10-10 PubMedCentralCrossRefPubMedGoogle Scholar
  48. 48.
    Sumida GM, Yamada S (2013) Self-contact elimination by membrane fusion. Proc Natl Acad Sci USA 110(47):18958–18963. doi: 10.1073/pnas.1311135110 PubMedCentralCrossRefPubMedGoogle Scholar
  49. 49.
    Moreno JL, Mikhailenko I, Tondravi MM, Keegan AD (2007) IL-4 promotes the formation of multinucleated giant cells from macrophage precursors by a STAT6-dependent, homotypic mechanism: contribution of E-cadherin. In: J Leukoc Biol, vol 82. United States, pp 1542–1553. doi: 10.1189/jlb.0107058
  50. 50.
    Helming L, Gordon S (2009) Molecular mediators of macrophage fusion. Trends Cell Biol 19:514–522. doi: 10.1016/j.tcb.2009.07.005 CrossRefPubMedGoogle Scholar
  51. 51.
    Helming L, Tomasello E, Kyriakides TR, Martinez FO, Takai T, Gordon S, Vivier E (2008) Essential role of DAP12 signaling in macrophage programming into a fusion-competent state. Sci Signal 1:ra11. doi: 10.1126/scisignal.1159665 PubMedCentralCrossRefPubMedGoogle Scholar
  52. 52.
    Shabo I, Olsson H, Stal O, Svanvik J (2013) Breast cancer expression of DAP12 is associated with skeletal and liver metastases and poor survival. Clin Breast Cancer 13(5):371–377. doi: 10.1016/j.clbc.2013.05.003 CrossRefPubMedGoogle Scholar
  53. 53.
    Xing Z, Jordana M, Kirpalani H, Driscoll KE, Schall TJ, Gauldie J (1994) Cytokine expression by neutrophils and macrophages in vivo: endotoxin induces tumor necrosis factor-alpha, macrophage inflammatory protein-2, interleukin-1 beta, and interleukin-6 but not RANTES or transforming growth factor-beta 1 mRNA expression in acute lung inflammation. Am J Respir Cell Mol Biol 10(2):148–153CrossRefPubMedGoogle Scholar
  54. 54.
    Wu Y, Zhou BP (2010) TNF-alpha/NF-kappaB/Snail pathway in cancer cell migration and invasion. In: Br J Cancer, vol 102. England, pp 639–644. doi: 10.1038/sj.bjc.6605530
  55. 55.
    Kim S, Choi JH, Kim JB, Nam SJ, Yang JH, Kim JH, Lee JE (2008) Berberine suppresses TNF-alpha-induced MMP-9 and cell invasion through inhibition of AP-1 activity in MDA-MB-231 human breast cancer cells. In: Molecules, vol 13. Switzerland, pp 2975–2985. doi: 10.3390/molecules13122975
  56. 56.
    Skokos EA, Charokopos A, Khan K, Wanjala J, Kyriakides TR (2011) Lack of TNF-alpha-induced MMP-9 production and abnormal E-cadherin redistribution associated with compromised fusion in MCP-1-null macrophages. In: Am J Pathol, vol 178, United States, pp 2311–2321. doi: 10.1016/j.ajpath.2011.01.045
  57. 57.
    Ma L, Lan F, Zheng Z, Xie F, Wang L, Liu W, Han J, Zheng F, Xie Y, Huang Q (2012) Epidermal growth factor (EGF) and interleukin (IL)-1beta synergistically promote ERK1/2-mediated invasive breast ductal cancer cell migration and invasion. In: Mol Cancer, vol 11, England, p 79. doi: 10.1186/1476-4598-11-79
  58. 58.
    Coussens LM, Werb Z (2002) Inflammation and cancer. Nature 420(6917):860–867PubMedCentralCrossRefPubMedGoogle Scholar
  59. 59.
    Eltzschig HK, Carmeliet P (2011) Hypoxia and inflammation. N Engl J Med 364(7):656–665. doi: 10.1056/NEJMra0910283 PubMedCentralCrossRefPubMedGoogle Scholar
  60. 60.
    Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related inflammation. Nature 454(7203):436–444. doi: 10.1038/nature07205 CrossRefPubMedGoogle Scholar
  61. 61.
    Tang ZN, Zhang F, Tang P, Qi XW, Jiang J (2011) Hypoxia induces RANK and RANKL expression by activating HIF-1alpha in breast cancer cells. In: Biochem Biophys Res Commun, vol 408, United States, pp 411–416. doi: 10.1016/j.bbrc.2011.04.035
  62. 62.
    Tang ZN, Zhang F, Tang P, Qi XW, Jiang J (2011) RANKL-induced migration of MDA-MB-231 human breast cancer cells via Src and MAPK activation. Oncol Rep 26(5):1243–1250. doi: 10.3892/or.2011.1368 PubMedGoogle Scholar
  63. 63.
    Yu M, Qi X, Moreno JL, Farber DL, Keegan AD (2011) NF-kappaB signaling participates in both RANKL- and IL-4-induced macrophage fusion: receptor cross-talk leads to alterations in NF-kappaB pathways. J Immunol, vol 187, United States, pp 1797–1806. doi: 10.4049/jimmunol.1002628

Copyright information

© Springer Basel 2015

Authors and Affiliations

  • Marieke Mohr
    • 1
  • Songül Tosun
    • 1
  • Wolfgang H. Arnold
    • 2
  • Frank Edenhofer
    • 3
  • Kurt S. Zänker
    • 1
  • Thomas Dittmar
    • 1
    Email author
  1. 1.Institute of Immunology and Experimental Oncology, Center for Biomedical Education and ResearchWitten/Herdecke UniversityWittenGermany
  2. 2.Department of Biological and Material Sciences in Dentistry, School of Dentistry, Faculty of HealthWitten/Herdecke UniversityWittenGermany
  3. 3.Stem Cell and Regenerative Medicine Group, Institute of Anatomy and Cell BiologyJulius-Maximilians-University WürzburgWürzburgGermany

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