Skip to main content

Advertisement

Log in

Developing in vitro models of human ductal carcinoma in situ from primary tissue explants

  • Preclinical study
  • Published:
Breast Cancer Research and Treatment Aims and scope Submit manuscript

Abstract

Because there are currently no reliable predictors for progression of ductal carcinoma in situ (DCIS) to invasive disease, nearly all patients receive comprehensive therapy, leading to over-treatment in many cases. Few in vitro models for studying DCIS progression have been developed. We report here the successful culture and expansion of primary DCIS from surgical specimens using a conditional reprogramming protocol. Patients with percutaneous core-needle biopsy demonstrating DCIS were enrolled in a tissue banking protocol after informed consent was received. Fresh tissue was taken from lumpectomy or mastectomy specimens, mechanically and enzymatically dissociated, cultured in medium conditioned by irradiated mouse fibroblasts and supplemented with rho-associated protein kinase (ROCK) inhibitor, and characterized by immunocytochemistry. Out of 33 DCIS cases, 58 % (19) were expanded for up to 2 months in culture, and 42 % (14) were frozen immediately after mechanical dissociation for future growth. The cultures are almost exclusively composed of cytokeratin 8- and EpCAM-positive luminal and cytokeratin 14-, cytokeratin 5-, and p63-positive basal mammary epithelial cells, suggesting maintenance of heterogeneity in vitro. Furthermore, as assessed by luminal and basal marker expression, these cells retain their cellular identities both in the “conditionally reprogrammed” proliferative state and after conditioned media and ROCK inhibitor withdrawal. When grown to 100 % confluency, the cultures organize into luminal and basal layers as well as luminal compartments surrounded by basal cells. Primary cultures of DCIS derived directly from patient tissues can be generated and may serve as in vitro models for the study of DCIS.

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

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Fonseca R, Hartmann LC, Petersen IA, Donohue JH, Crotty TB, Gisvold JJ (1997) Ductal carcinoma in situ of the breast. Ann Intern Med 127(11):1013–1022

    Article  CAS  PubMed  Google Scholar 

  2. Mukhtar RA, Wong JM, Esserman LJ (2015) Preventing Overdiagnosis and overtreatment: just the next step in the evolution of breast cancer care. J Nat Compr Cancer Netw 13(6):737–743

    Google Scholar 

  3. Sanders ME, Schuyler PA, Dupont WD, Page DL (2005) The natural history of low-grade ductal carcinoma in situ of the breast in women treated by biopsy only revealed over 30 years of long-term follow-up. Cancer 103(12):2481–2484. doi:10.1002/cncr.21069

    Article  PubMed  Google Scholar 

  4. Sanders ME, Schuyler PA, Simpson JF, Page DL, Dupont WD (2015) Continued observation of the natural history of low-grade ductal carcinoma in situ reaffirms proclivity for local recurrence even after more than 30 years of follow-up. Mod Pathol 28(5):662–669. doi:10.1038/modpathol.2014.141

    Article  PubMed  Google Scholar 

  5. Erbas B, Provenzano E, Armes J, Gertig D (2006) The natural history of ductal carcinoma in situ of the breast: a review. Breast Cancer Res Treat 97(2):135–144. doi:10.1007/s10549-005-9101-z

    Article  PubMed  Google Scholar 

  6. Silverstein MJ, Lagios MD (2015) Treatment selection for patients with ductal carcinoma in situ (DCIS) of the breast using the University of Southern California/Van Nuys (USC/VNPI) prognostic index. Breast J 21(2):127–132. doi:10.1111/tbj.12368

    Article  PubMed  Google Scholar 

  7. Solin LJ, Gray R, Baehner FL, Butler SM, Hughes LL, Yoshizawa C, Cherbavaz DB, Shak S, Page DL, Sledge GW Jr, Davidson NE, Ingle JN, Perez EA, Wood WC, Sparano JA, Badve S (2013) A multigene expression assay to predict local recurrence risk for ductal carcinoma in situ of the breast. J Natl Cancer Inst 105(10):701–710. doi:10.1093/jnci/djt067

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Rakovitch E, Nofech-Mozes S, Hanna W, Baehner FL, Saskin R, Butler SM, Tuck A, Sengupta S, Elavathil L, Jani PA, Bonin M, Chang MC, Robertson SJ, Slodkowska E, Fong C, Anderson JM, Jamshidian F, Miller DP, Cherbavaz DB, Shak S, Paszat L (2015) A population-based validation study of the DCIS Score predicting recurrence risk in individuals treated by breast-conserving surgery alone. Breast Cancer Res Treat 152(2):389–398. doi:10.1007/s10549-015-3464-6

    Article  PubMed Central  PubMed  Google Scholar 

  9. Esserman LJ, Thompson IM Jr, Reid B (2013) Overdiagnosis and overtreatment in cancer: an opportunity for improvement. JAMA 310(8):797–798. doi:10.1001/jama.2013.108415

    Article  CAS  PubMed  Google Scholar 

  10. Barnabas N, Cohen D (2013) Phenotypic and molecular characterization of MCF10DCIS and SUM breast cancer cell lines. Int J Breast Cancer 2013:872743. doi:10.1155/2013/872743

    Article  PubMed Central  PubMed  Google Scholar 

  11. Kalaany NY, Sabatini DM (2009) Tumours with PI3 K activation are resistant to dietary restriction. Nature 458(7239):725–731. doi:10.1038/nature07782

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Miller FR, Santner SJ, Tait L, Dawson PJ (2000) MCF10DCIS.com xenograft model of human comedo ductal carcinoma in situ. J Natl Cancer Inst 92(14):1185–1186

    Article  CAS  PubMed  Google Scholar 

  13. Forozan F, Veldman R, Ammerman CA, Parsa NZ, Kallioniemi A, Kallioniemi OP, Ethier SP (1999) Molecular cytogenetic analysis of 11 new breast cancer cell lines. Br J Cancer 81(8):1328–1334. doi:10.1038/sj.bjc.6695007

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. Gupta PB, Kuperwasser C (2004) Disease models of breast cancer. Drug Discov Today 1(1):9–16. doi:10.1016/j.ddmod.2004.05.001

    CAS  Google Scholar 

  15. Behbod F, Kittrell FS, LaMarca H, Edwards D, Kerbawy S, Heestand JC, Young E, Mukhopadhyay P, Yeh HW, Allred DC, Hu M, Polyak K, Rosen JM, Medina D (2009) An intraductal human-in-mouse transplantation model mimics the subtypes of ductal carcinoma in situ. Breast Cancer Res 11(5):R66. doi:10.1186/bcr2358

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Valdez KE, Fan F, Smith W, Allred DC, Medina D, Behbod F (2011) Human primary ductal carcinoma in situ (DCIS) subtype-specific pathology is preserved in a mouse intraductal (MIND) xenograft model. J Pathol 225(4):565–573. doi:10.1002/path.2969

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Leal CB, Schmitt FC, Bento MJ, Maia NC, Lopes CS (1995) Ductal carcinoma in situ of the breast. Histologic categorization and its relationship to ploidy and immunohistochemical expression of hormone receptors, p53, and c-erbB-2 protein. Cancer 75(8):2123–2131

    Article  CAS  PubMed  Google Scholar 

  18. Collins LC, Schnitt SJ (2005) HER2 protein overexpression in estrogen receptor-positive ductal carcinoma in situ of the breast: frequency and implications for tamoxifen therapy. Mod Pathol 18(5):615–620. doi:10.1038/modpathol.3800360

    Article  CAS  PubMed  Google Scholar 

  19. Liu X, Ory V, Chapman S, Yuan H, Albanese C, Kallakury B, Timofeeva OA, Nealon C, Dakic A, Simic V, Haddad BR, Rhim JS, Dritschilo A, Riegel A, McBride A, Schlegel R (2012) ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells. Am J Pathol 180(2):599–607. doi:10.1016/j.ajpath.2011.10.036

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Palechor-Ceron N, Suprynowicz FA, Upadhyay G, Dakic A, Minas T, Simic V, Johnson M, Albanese C, Schlegel R, Liu X (2013) Radiation induces diffusible feeder cell factor(s) that cooperate with ROCK inhibitor to conditionally reprogram and immortalize epithelial cells. Am J Pathol 183(6):1862–1870. doi:10.1016/j.ajpath.2013.08.009

    Article  CAS  PubMed  Google Scholar 

  21. Saenz FR, Ory V, AlOtaiby M, Rosenfield S, Furlong M, Cavalli LR, Johnson MD, Liu X, Schlegel R, Wellstein A, Riegel AT (2014) Conditionally reprogrammed normal and transformed mouse mammary epithelial cells display a progenitor-cell-like phenotype. PLoS One 9(5):e97666. doi:10.1371/journal.pone.0097666

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Suprynowicz FA, Upadhyay G, Krawczyk E, Kramer SC, Hebert JD, Liu X, Yuan H, Cheluvaraju C, Clapp PW, Boucher RC Jr, Kamonjoh CM, Randell SH, Schlegel R (2012) Conditionally reprogrammed cells represent a stem-like state of adult epithelial cells. Proc Natl Acad Sci USA 109(49):20035–20040. doi:10.1073/pnas.1213241109

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  23. Brimo F, Aprikian A, Latour M, Tetu B, Doueik A, Scarlata E, Hamel L, McKercher G, Saad F, Lacombe L, Carmel M, Chevalier S (2013) Strategies for biochemical and pathologic quality assurance in a large multi-institutional biorepository; The experience of the PROCURE Quebec Prostate Cancer Biobank. Biopreserv Biobank 11(5):285–290. doi:10.1089/bio.2013.0025

    Article  PubMed Central  PubMed  Google Scholar 

  24. Brimo F, Sircar K, Chevalier S, Saad F, Lacombe L, Tetu B, Scarlata E, Aprikian A (2012) Banking of fresh-frozen prostate tissue using the alternate mirror image protocol: methods, validation, and impact on the pathological prognostic parameters in radical prostatectomy. Cell Tissue Bank 13(4):631–638. doi:10.1007/s10561-011-9284-y

    Article  PubMed  Google Scholar 

  25. Novaro V, Roskelley CD, Bissell MJ (2003) Collagen-IV and laminin-1 regulate estrogen receptor alpha expression and function in mouse mammary epithelial cells. J Cell Sci 116(Pt 14):2975–2986. doi:10.1242/jcs.00523

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  26. Anderson E, Clarke RB, Howell A (1998) Estrogen responsiveness and control of normal human breast proliferation. J Mammary Gland Biol Neoplas 3(1):23–35

    Article  CAS  Google Scholar 

  27. Ronnov-Jessen L, Petersen OW, Bissell MJ (1996) Cellular changes involved in conversion of normal to malignant breast: importance of the stromal reaction. Physiol Rev 76(1):69–125

    CAS  PubMed  Google Scholar 

  28. Taylor-Papadimitriou J, Stampfer M, Bartek J, Lewis A, Boshell M, Lane EB, Leigh IM (1989) Keratin expression in human mammary epithelial cells cultured from normal and malignant tissue: relation to in vivo phenotypes and influence of medium. J Cell Sci 94(Pt 3):403–413

    PubMed  Google Scholar 

  29. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, Clark L, Bayani N, Coppe JP, Tong F, Speed T, Spellman PT, DeVries S, Lapuk A, Wang NJ, Kuo WL, Stilwell JL, Pinkel D, Albertson DG, Waldman FM, McCormick F, Dickson RB, Johnson MD, Lippman M, Ethier S, Gazdar A, Gray JW (2006) A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell 10(6):515–527. doi:10.1016/j.ccr.2006.10.008

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Charafe-Jauffret E, Ginestier C, Monville F, Finetti P, Adelaide J, Cervera N, Fekairi S, Xerri L, Jacquemier J, Birnbaum D, Bertucci F (2006) Gene expression profiling of breast cell lines identifies potential new basal markers. Oncogene 25(15):2273–2284. doi:10.1038/sj.onc.1209254

    Article  CAS  PubMed  Google Scholar 

  31. Cheung KJ, Gabrielson E, Werb Z, Ewald AJ (2013) Collective invasion in breast cancer requires a conserved basal epithelial program. Cell 155(7):1639–1651. doi:10.1016/j.cell.2013.11.029

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  32. Yang S, Kim HM (2014) ROCK inhibition activates MCF-7 cells. PLoS One 9(2):e88489. doi:10.1371/journal.pone.0088489

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  33. Yong JW, Choong ML, Wang S, Wang Y, Lim SQ, Lee MA (2014) Characterization of ductal carcinoma in situ cell lines established from breast tumor of a Singapore Chinese patient. Cancer Cell Int 14(1):94. doi:10.1186/s12935-014-0094-8

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  34. Labarge MA, Garbe JC, Stampfer MR (2013) Processing of human reduction mammoplasty and mastectomy tissues for cell culture. J Vis Exp. doi:10.3791/50011

    PubMed Central  PubMed  Google Scholar 

  35. Romanov SR, Kozakiewicz BK, Holst CR, Stampfer MR, Haupt LM, Tlsty TD (2001) Normal human mammary epithelial cells spontaneously escape senescence and acquire genomic changes. Nature 409(6820):633–637. doi:10.1038/35054579

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The cytogenetic studies were carried out in the UPCI Cell Culture and Cytogenetics Facility, supported in part by the UPCI and by award P30CA047904. We gratefully acknowledge the support of pilot funds from the Women’s Cancer Research Center of UPCI, the Shear Women’s Cancer and Personalized Medicine Research Fund. We would also like to acknowledge technical assistance from Chandra Bathula, PhD at the University of Pittsburgh Cancer Institute and valuable advice from Harikrishna Nakshatri, PhD at Indiana University.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Priscilla F. McAuliffe.

Ethics declarations

Conflict of Interest

The authors declare that they have no conflicts of interest.

Ethical Approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the University of Pittsburgh Institutional Review Board and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. All patients signed informed consent prior to participation. The authors declare that all experiments comply with the current laws of the United States of America.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Brown, D.D., Dabbs, D.J., Lee, A.V. et al. Developing in vitro models of human ductal carcinoma in situ from primary tissue explants. Breast Cancer Res Treat 153, 311–321 (2015). https://doi.org/10.1007/s10549-015-3551-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10549-015-3551-8

Keywords

Navigation