Skip to main content

Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting

In Vitro Cellular & Developmental Biology - Animal volume 34, pages 711–721 (1998)Cite this article

Summary

Lineage analysis in vitro of heterogeneous tissues such as mammary epithelium requires the separation of constituent cell types and their growth as clones. The separation of virgin mouse mammary luminal epithelial and myoepithelial cells by fluorescence-activated cell-sorting, their growth at clonal density, and the phenotyping of the clones obtained with cell-type specific markers are described in this paper. Epithelial cells were isolated by collagenase digestion followed by trypsinization, and the luminal and myoepithelial cells were flow-sorted with the rat monoclonal antibodies 33A10 and JB6, respectively. Sorted cells were cloned under, using low oxygen conditions (<5% vol/vol), in medium containing cholera toxin and insulin, with an irradiated feeder layer of 3T3-L1 cells. Clones were characterized morphologically, and antigenically by multiple immunofluorescence with a panel of antibodies to cytoskeletal antigens specific to either luminal epithelial or myoepithelial cells in situ. Whereas sorted myoepithelial cells gave a single clone type, sorted luminal cells gave three morphological clone types, two of which grew rapidly. All myoepithelially derived clones showed a limited proliferative capacity in vitro, in contrast to their rat and human counterparts, as shown in previous studies. The present results with sorted mouse cells have also allowed the stability of the differentiated phenotype in mouse, rat, and human mammary luminal epithelial and myoepithelial cells in primary clonal culture to be compared. They show that the mouse mammary cells are the least stable in terms of expression of differentiation-specific cytoskeletal markers in vitro.

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

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

References

  1. Asch, H. L.; Asch, B. B. Expression of keratins and other cytoskeletal proteins in mouse mammary epithelium during the normal developmental cycle and primary culture. Dev. Biol. 107:470–482; 1985.

    PubMed  Article  CAS  Google Scholar 

  2. Bartek, J.; Taylor-Papadimitriou, J.; Miller, N., et al. Patterns of expression of keratin 19 as detected with monoclonal antibodies in human breast tissues and tumours. Int. J. Cancer 36:299–306; 1985.

    PubMed  CAS  Google Scholar 

  3. Clarke, C.; Titley, J.; Davies, S., et al. An immunomagnetic separation method using superparamagnetic (MACS) beads for large-scale purification of human mammary luminal and myoepithelial cells. Epithelial Cell Biol. 3:38–46; 1994.

    PubMed  CAS  Google Scholar 

  4. Das, N. K.; Hosick, H. L.; Nandi, S. Influence of seeding density on multicellular organization and nuclear events in cultures of normal and neoplastic mouse mammary epithelium. J. Natl. Cancer Inst. 52:849–861; 1974.

    PubMed  CAS  Google Scholar 

  5. Dundas, S. D.; Ormerod, M. G.; Gusterson, B. A., et al. Characterization of luminal and basal cells flow-sorted from the adult rat mammary parenchyma. J. Cell Sci. 100:459–471; 1991.

    PubMed  Google Scholar 

  6. DeOme, K. B.; Faulklin, L. J.; Bern, H. A., et al. Development of mammary tumors from hyperplastic alveolar nodules transplanted into gland-free mammary fat pads of female C3H mice. Cancer Res. 19:515–520; 1959.

    PubMed  CAS  Google Scholar 

  7. Edwards, P. W. E. Tissue reconstitution models of breast cancer. Cancer Surv. 16:79–96; 1993.

    PubMed  CAS  Google Scholar 

  8. Ehmann, U. K.; Peterson, W. D.; Misfeldt, D. S. To grow mouse mammary epithelial cells in culture. J. Cell Biol. 98:1026–1032; 1984.

    PubMed  Article  CAS  Google Scholar 

  9. Emerman, J. T.; Vogl. A. W. Cell size and shape changes in the myoepithelium of the mammary gland during differentiation. Anat. Rec. 216:405–415; 1986.

    PubMed  Article  CAS  Google Scholar 

  10. Joshi, K.; Ellis, J. T.; Hughes, C. M., et al. Cellular proliferation in the rat mammary gland during pregnancy and lactation. Lab. Invest. 54:52–61; 1986.

    PubMed  CAS  Google Scholar 

  11. Joshi, K.; Smith, J. A.; Perusinghe, N., et al. Cell proliferation in the human mammary epithelium. Differential contribution by epithelial and myoepithelial cells. Am. J. Pathol. 124:199–206; 1986.

    PubMed  CAS  Google Scholar 

  12. Lane, E. B. Monoclonal antibodies provide specific intramolecular markers for the study of epithelial tonofilament organization. J. Cell Biol. 92:665–673; 1982.

    PubMed  Article  CAS  Google Scholar 

  13. Lane, E. B.; Bartek, J.; Purkis, P. E., et al. Keratin antigens in differentiating skin. Ann. NY Acad. Sci. 455:241–258; 1985.

    PubMed  Article  CAS  Google Scholar 

  14. Lin, T. P.; Hom, Y. K.; Richards, J., et al. Effects of antioxidants and reduced oxygen tension on rat mammary epithelial cells in culture. In Vitro Cell. Dev. Biol. 27A:191–196; 1991.

    PubMed  Article  CAS  Google Scholar 

  15. Lin, C. Q.; Dempsey, P. J.; Coffey, R. J., et al. Extracellular matrix regulates whey acidic protein gene expression by suppression of TGF-alpha in mouse mammary epithelial cells: studies in culture and in transgenic mice. J. Cell Biol. 129:1115–1126; 1995.

    PubMed  Article  CAS  Google Scholar 

  16. Medina, D.; Oborn, C. J.; Kittrell, F. S., et al. Properties of mouse mammary epithelial cell lines characterized by in vivo transplantation and in vitro immunocytochemical methods. J. Natl. Cancer Inst. 76:1143–1156; 1986.

    PubMed  CAS  Google Scholar 

  17. Moll, R.; Franke, W. W.; Schiller, D. L., et al. The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11–24; 1982.

    PubMed  Article  CAS  Google Scholar 

  18. O’Hare, M. J.; Ormerod, M. G.; Monaghan, P., et al. Characterization in vitro of luminal and myoepithelial cells isolated from the human mammary gland by cell sorting. Differentiation 46:209–221; 1991.

    PubMed  Article  CAS  Google Scholar 

  19. Pierce, D. F.; Gorska, A. E.; Chytil, A., et al. Mammary tumor suppression by transforming growth factor beta 1 transgene expression. Proc. Natl. Acad. Sci. USA 92:4254–4258; 1995.

    PubMed  Article  CAS  Google Scholar 

  20. Purkis, P. E.; Steel, J. B.; Mackenzie, I. C., et al. Antibody markers of basal cells in complex epithelia. J. Cell Sci. 97:39–50; 1990.

    PubMed  Google Scholar 

  21. Reichmann, E.; Ball, R.; Groner, B., et al. New mammary epithelial and fibroblastic cell clones in coculture form structures competent to differentiate functionally. J. Cell Biol. 108:1127–1138; 1989.

    PubMed  Article  CAS  Google Scholar 

  22. Russo, I.; Russo, J. H. Biological and molecular bases of mammary carcinogenesis. Lab. Invest. 57:112–137; 1987.

    PubMed  CAS  Google Scholar 

  23. Russo, J.; Gusterson, B. A.; Rogers, A. E., et al. Comparative study of human and rat mammary tumorigenesis. Lab. Invest. 62:244–278; 1990.

    PubMed  CAS  Google Scholar 

  24. Sapino, A.; Macri, L.; Gugliotta, P., et al. Immunocytochemical identification of proliferating cell types in mouse mammary gland. J. Histochem. Cytochem. 38:1541–1547; 1990.

    PubMed  CAS  Google Scholar 

  25. Sapino, A.; Macri, L.; Tonda, L., et al. Oxytocin enhances myoepithelial cell differentiation and proliferation in the mouse mammary gland. Endocrinology 133:838–842; 1993.

    PubMed  Article  CAS  Google Scholar 

  26. Schuuring, E.; van der Leede, B. J.; Willems, R., et al. Differentiation-dependent expression of provirus-activated int-1 oncogene in clonal cell lines derived from a mouse mammary tumor. Oncogene 5:459–465; 1990.

    PubMed  CAS  Google Scholar 

  27. Sekhri, K. K.; Pitelka, D. R.; DeOme, K. B. Studies of mouse mammary glands. I. Cytomorphology of the normal mammary gland. J. Natl. Cancer Inst. 39:459–490; 1967.

    PubMed  CAS  Google Scholar 

  28. Skalli, O.; Ropraz, P.; Trzeciak, A., et al. A monoclonal antibody against α-smooth muscle actin: a new probe for smooth muscle differentiation. J. Cell Biol. 103:2787–2796; 1986.

    PubMed  Article  CAS  Google Scholar 

  29. Soloff, M. S.; Chakraborty, J.; Sadhukhan, P., et al. Purification and characterization of mammary myoepithelial and secretory cells from the lactating rat. Endocrinology 106:887–897; 1980.

    PubMed  CAS  Article  Google Scholar 

  30. Sonnenberg, A.; Daams, H.; Calafat, J., et al. In vitro differentiation and progression of mouse mammary tumor cells. Cancer Res. 46:5913–5922; 1986.

    PubMed  CAS  Google Scholar 

  31. Sonnenberg, A.; Daams, H.; van der Valk, M. A., et al. Development of mouse mammary gland: identification of stages in differentiation of luminal and myoepithelial cells using monoclonal antibodies and polyvalent antiserum against keratin. J. Histochem. Cytochem. 34:1037–1046; 1986.

    PubMed  CAS  Google Scholar 

  32. Streuli, C. H.; Bissell, M. J. Expression of extracellular matrix components is regulated by substratum. J. Cell Biol. 110:1404–1415; 1990.

    Google Scholar 

  33. Troyanovsky, S. M.; Guelstein, V. I.; Tchipysheva, T. A., et al. Patterns of expression of keratin 17 in human epithelia: dependency on cell position. J. Cell Sci. 93:419–426; 1989.

    PubMed  Google Scholar 

  34. Vonderhaar, B. K. Regulation of development of the normal mammary gland by hormones and growth factors. Cancer Res. Treatment 40:251–266; 1988.

    CAS  Google Scholar 

  35. Webster, M. A.; Muller, W. J. Mammary tumorigenesis and metastasis in transgenic mice. Semin. Cancer Biol. 5:69–76; 1994.

    PubMed  CAS  Google Scholar 

  36. Wetzels, R. H.; Kuijpers, H. J. H.; Lane, E. B., et al. Basal cell specific and hyperproliferation-related keratins in human breast cancer. Am. J. Pathol. 138:751–763; 1991.

    PubMed  CAS  Google Scholar 

Download references

Author information

Affiliations

  1. Section of Cell Biology and Experimental Pathology, Institute of Cancer Research: Royal Cancer Hospital, SM2 5NG, Sutton, Surrey, United Kingdom

    Matthew J. Smalley & Jenny Titley

  2. LICR/UCL Breast Cancer Laboratory, Department of Surgery, University College London, Charles Bell House, 67-73 Riding House Street, W1P 7LD, London, United Kingdom

    Michael J. O’Hare

Authors
  1. Matthew J. Smalley
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jenny Titley
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michael J. O’Hare
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Smalley, M.J., Titley, J. & O’Hare, M.J. Clonal characterization of mouse mammary luminal epithelial and myoepithelial cells separated by fluorescence-activated cell sorting. In Vitro Cell.Dev.Biol.-Animal 34, 711–721 (1998). https://doi.org/10.1007/s11626-998-0067-0

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11626-998-0067-0

Key words

  • epithelium
  • murine
  • cloning
  • cytokeratins
  • oxygen
  • growth