Journal of Cell Communication and Signaling

, Volume 13, Issue 1, pp 129–143 | Cite as

A reproducible scaffold-free 3D organoid model to study neoplastic progression in breast cancer

  • Sabra I. Djomehri
  • Boris Burman
  • Maria E. Gonzalez
  • Shuichi TakayamaEmail author
  • Celina G. KleerEmail author
Nuts and Bolts


While 3D cellular models are useful to study biological processes, gel-embedded organoids have large variability. This paper describes high-yield production of large (~1 mm diameter), scaffold-free, highly-spherical organoids in a one drop-one organoid format using MCF10A cells, a non-tumorigenic breast cell line. These organoids display a hollow lumen and secondary acini, and express mammary gland-specific and progenitor markers, resembling normal human breast acini. When subjected to treatment with TGF-β, the hypoxia-mimetic reagent CoCl2, or co-culture with mesenchymal stem/stromal cells (MSC), the organoids increase collagen I production and undergo large phenotypic and morphological changes of neoplastic progression, which were reproducible and quantifiable. Advantages of this scaffold-free, 3D breast organoid model include high consistency and reproducibility, ability to measure cellular collagen I production without noise from exogenous collagen, and capacity to subject the organoid to various stimuli from the microenvironment and exogenous treatments with precise timing without concern of matrix binding. Using this system, we generated organoids from primary metaplastic mammary carcinomas of MMTV-Cre;Ccn6fl/fl mice, which retained the high grade spindle cell morphology of the primary tumors. The platform is envisioned to be useful as a standardized 3D cellular model to study how microenvironmental factors influence breast tumorigenesis, and to potential therapeutics.


3D cell culture Organoids Hanging drop Human breast cancer Morphogenesis Phenotype Microenvironment EMT Mesenchymal stromal cells Metaplastic CCN6 



We thank Tina Fields (Research Histology and IHC Laboratory, Rogel Cancer Center, University of Michigan) and Dafydd Thomas (Department of Pathology, Michigan Medicine) for their assistance with sectioning, immunohistochemistry, and immunostaining procedures and Dr. Brendan Leung for early studies. The study was supported by R01 grants to Dr. Celina Kleer (R01 CA107469, R01 CA125577, and the Karlene Kulp Fund Judy & Ken Robinson Fund), Dr. Shuichi Takayama (R01 CA196018), and the University of Michigan Rogel Cancer Center support grant P30CA046592. The authors would also like to thank Dr. Alexey Nesvizhskii for continued support by the Proteome Informatics of Cancer Training Program (PICTP) (NIH 5T32CA140044-08).

Author contributions

S.D., C.K., and S.T. were involved in the study conception, design, analyses and interpretation. S.D. performed data acquisition and analysis, and B.B. and M.M. helped with completion of experiments and interpretation. S.D. prepared the manuscript and figures, C.K. and S.T. helped with the revision of the manuscript. All authors read and approved the final manuscript.

Compliance with ethical standards

Competing financial interests

The authors declare no competing financial interests.

Supplementary material

12079_2018_498_MOESM1_ESM.pptx (4.3 mb)
Supplemental Figure 1 Spheroid optimization assay at varying Matrigel concentrations (0%, 1%, 1.5% and 2% v/v) from time = 0 to 72 h (N = 25 per subgroup) indicated by A) representative brightfield images, B) average spheroid diameter, C) percentage of droplets containing multi-spheres per droplet, called “loose cell aggregates”, and D) percentage of droplets that form one sphere per droplet. Scalebar = 100 μm. (PPTX 4365 kb)
12079_2018_498_MOESM2_ESM.pptx (72 kb)
Supplemental Figure 2 Comparison of stem cell vs differentiation marker expression for western blot analysis of MCF10A cells cultured in monolayer and 3D at days 4 and 8, where “MG” identifies MCF10A spheroids seeded without Matrigel. MDA-MB-231 cells cultured in 2D were shown as a control. Antibodies used: E-cadherin, Vimentin, ALDH1, CD44, CD49f, EpCam, and β-actin. Values above E-cadherin blot shows densitometric analysis of relative concentrations. (PPTX 72 kb)
12079_2018_498_MOESM3_ESM.pptx (16.6 mb)
Supplemental Figure 3 Summary of organoids developed in hanging drop with A) MCF10A vs MDA-MB-231 organoids at days 4, 8, and 16 with a comparison of monolayer phase contrast images, H&E staining, and confocal imaging with DAPI, CK5/6, and CK18 status (expression results merged), B) comparison of H&E and collagen I staining from MCF10A, TGFβ1, CoCl2, and co-culture organoids at days 8, 12, and 16. Scale bar =200 μm. (PPTX 16956 kb)


  1. Abd El-Rehim DM et al (2004) Expression of luminal and basal cytokeratins in human breast carcinoma. J Pathol 203:661–671Google Scholar
  2. Aijian AP, Garrell RL (2015) Digital microfluidics for automated hanging drop cell spheroid culture. J Lab Autom 20:283–295Google Scholar
  3. Anwar TE, Kleer CG (2013) Tissue-based identification of stem cells and epithelial-to- mesenchymal transition in breast cancer. Hum Pathol 44:1457–1464PubMedCentralGoogle Scholar
  4. Barcellos-Hoff MH, Aggeler J, Ram TG, Bissell MJ (1989) Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 105:223–235PubMedCentralGoogle Scholar
  5. Bissell MJ, Hall HG, Parry G (1982) How does the extracellular matrix direct gene expression. J Theor Biol 99:31–68Google Scholar
  6. Boecker W, Buerger H (2003) Evidence of progenitor cells of glandular and myoepithelial cell lineages in the human adult female breast epithelium: a new progenitor (adult stem) cell concept. Cell Prolif 36(Suppl 1):73–84Google Scholar
  7. Celli JP et al (2014) An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models. Sci Rep 4:1–10Google Scholar
  8. Cerchiari AE et al (2015) A strategy for tissue self-organization that is robust to cellular heterogeneity and plasticity. Proc Natl Acad Sci 112:2287–2292Google Scholar
  9. Chandler EM et al (2011) Stiffness of photocrosslinked RGD-alginate gels regulates adipose progenitor cell behavior. Biotechnol Bioeng 108:1683–1692Google Scholar
  10. Chung W et al (2017) Single-cell RNA-seq enables comprehensive tumour and immune cell profiling in primary breast cancer. Nat Commun 8Google Scholar
  11. Debnath J, Muthuswamy SK, Brugge JS (2003) Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30:256–268Google Scholar
  12. Emerman J, Pitelka DR (1977) Maintenance and induction of morphological differentiation in dissociated mammary epithelium on floating collagen membranes. In Vitro 13:316–328Google Scholar
  13. Fata JE et al (2007) The MAPKERK-1,2pathway integrates distinct and antagonistic signals from TGFα and FGF7 in morphogenesis of mouse mammary epithelium. Dev Biol 306:193–207PubMedCentralGoogle Scholar
  14. Gaiko-Shcherbak A et al (2015) The acinar cage: basement membranes determine molecule exchange and mechanical stability of human breast cell acini. PLoS One 10:1–20Google Scholar
  15. Gonzalez ME et al (2017) Mesenchymal stem cell-induced DDR2 mediates stromal-breast Cancer interactions and metastasis growth. Cell Rep 18:1215–1228PubMedCentralGoogle Scholar
  16. Gusterson BA, Ross DT, Heath VJ, Stein T (2005) Basal cytokeratins and their relationship to the cellular origin and functional classification of breast cancer. Breast Cancer Res 7:143–148PubMedCentralGoogle Scholar
  17. Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK (2003) Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83:173–180Google Scholar
  18. Klos KS et al (2014) Building bridges toward invasion: tumor promoter treatment induces a novel protein kinase C-dependent phenotype in MCF10A mammary cell acini. PLoS One 9:1–11Google Scholar
  19. Lance A et al (2016) Increased extracellular matrix density decreases MCF10A breast cell acinus formation in 3D culture conditions. J Tissue Eng Regen Med 10:71–80Google Scholar
  20. Lee GY, Kenny P a, Lee EH, Bissell MJ (2007) Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods 4:359–365PubMedCentralGoogle Scholar
  21. Lesher-Pérez SC et al (2017) Dispersible oxygen microsensors map oxygen gradients in three-dimensional cell cultures. Biomater. Sci. 5:2106–2113PubMedCentralGoogle Scholar
  22. Leung BM, Lesher-Perez SC, Matsuoka T, Moraes C, Takayama S (2015) Media additives to promote spheroid circularity and compactness in hanging drop platform. Biomater Sci 3:336–344Google Scholar
  23. Linnemann JR et al (2015) Quantification of regenerative potential in primary human mammary epithelial cells. Development 142:3239–3251PubMedCentralGoogle Scholar
  24. Mani SA et al (2008) The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133:704–715PubMedCentralGoogle Scholar
  25. Marina S, Bissell MJO (2017) A historical perspective of thinkin in three dimensions. J Cell Biol 216:1–10. Google Scholar
  26. Martin EE, Huang W, Anwar T, Arellano-Garcia C, Burman B, Guan J-L, Gonzalez ME, K CG (2017) MMTV-cre;Ccn6 knockout mice develop tumors recapitulating human metaplastic breast carcinomas. Oncogene 36:2275–2285Google Scholar
  27. Mueller S, Millonig G, G W (2009) The GOX/CAT system: a novel enzymatic method to independently control hydrogen peroxide and hypoxia in cell culture. Adv Med Sci 54:121–135Google Scholar
  28. Naba A, Hoersch S, Hynes RO (2012) Towards definition of an ECM parts list: an advance on GO categories. Matrix Biol 31:371–372PubMedCentralGoogle Scholar
  29. Naber HPH, Wiercinska E, ten Dijke P, van Laar T (2011) Spheroid assay to measure TGF-β-induced invasion. J Vis Exp:e3337.
  30. Nakano T et al (2012) Self-formation of optic cups and storable stratified neural retina from human ESCs. Cell Stem Cell 10:771–785Google Scholar
  31. Pal A, Kleer CG (2014) Three dimensional cultures: A tool to study normal acinar architecture vs. malignant transformation of breast cells. J Vis Exp:e51311.
  32. Pal A, Huang W, Toy KA, Kleer CG (2012) CCN6 knockdown disrupts acinar Organization of Breast Cells in three-dimensional cultures through up-regulation of type III TGF-β receptor. Neoplasia 14:1067–1074PubMedCentralGoogle Scholar
  33. Petersen OW, Ronnov-Jessen L, Howlett AR, Bissell MJ (1992) Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc Natl Acad Sci 89:9064–9068Google Scholar
  34. Qu Y et al (2015) Evaluation of MCF10A as a reliable model for normal human mammary epithelial cells. PLoS One 10:1–16Google Scholar
  35. Quadrato G et al (2017) Cell diversity and network dynamics in photosensitive human brain organoids. Nature 545:48–53PubMedCentralGoogle Scholar
  36. Reid JA, Mollica PM, Bruno RD, Sachs PC (2018) Consistent and reproducible cultures of large-scale 3D mammary epithelial structures using an accessible bioprinting platform. Breast Cancer Res 20:1–13. Google Scholar
  37. Rizwan A et al (2015) Metastatic breast cancer cells in lymph nodes increase nodal collagen density. Sci Rep 5:1–6Google Scholar
  38. Roerdink J, Meijster A (2000) The watershed transforms: definitions, algorithms and parallelization strategies. Fundam Inform 41(1-2):187–228.
  39. Sachs N et al (2018) A living biobank of breast Cancer organoids captures disease heterogeneity. Cell 172:373–386.e10Google Scholar
  40. Sato T et al (2011) Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141:1762–1772Google Scholar
  41. Simian M et al (2001) The interplay of matrix metalloproteinases, morphogens and growth factors is necessary for branching of mammary epithelial cells. Development 128:3117–3131PubMedCentralGoogle Scholar
  42. Thoma CR et al (2013) A high-throughput-compatible 3D microtissue co-culture system for phenotypic RNAi screening applications. J Biomol Screen 18:1330–1337Google Scholar
  43. Thoma CR, Zimmermann M, Agarkova I, Kelm JM, Krek W (2014) 3D cell culture systems modeling tumor growth determinants in cancer target discovery. Adv Drug Deliv Rev 69–70:29–41Google Scholar
  44. Tran DD, Corsa CAS, Biswas H, Aft RL, Longmore GD (2011) Temporal and spatial cooperation of Snail1 and Twist1 during epithelial-mesenchymal transition predicts for human breast Cancer recurrence. Mol Cancer Res 9:1644–1657PubMedCentralGoogle Scholar
  45. Tung Y-C et al (2011) High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst 136:473–478Google Scholar
  46. Vaapil M et al (2012) Hypoxic conditions induce a Cancer-like phenotype in human breast epithelial cells. PLoS One 7:e46543PubMedCentralGoogle Scholar
  47. Venugopalan G et al (2014) Multicellular architecture of malignant breast epithelia influences mechanics. PLoS One 9:e101955PubMedCentralGoogle Scholar
  48. Vidi P, Bissell MJ, Lelièvre SA (2013) Epithelial Cell Culture Protocols 945:193–219Google Scholar
  49. Vinci M et al (2012) Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biol 10:29PubMedCentralGoogle Scholar
  50. Wang M et al (2017) Role of tumor microenvironment in tumorigenesis. J Cancer 8:761–773PubMedCentralGoogle Scholar
  51. Weeber F, Ooft SN, Dijkstra KK, Voest EE (2017) Tumor organoids as a pre-clinical Cancer model for drug discovery. Cell Chem Biol 24:1092–1100Google Scholar
  52. Wellings SR (1980) A hypothesis of the origin of human breast cancer from the terminal ductal lobular unit. Pathol Res Pract 166:515–535Google Scholar
  53. Wetering M, De V et al (2015) Prospective derivation of a living organoid biobank of colorectal Cancer patients resource prospective derivation of a living organoid biobank of colorectal Cancer patients. Cell 161:933–945Google Scholar
  54. Wu D, Yotnda P (2011) Induction and testing of hypoxia in cell culture. J Vis Exp:4–7.
  55. Yin X et al (2016) Cell stem cell engineering stem cell organoids. Stem Cell 18:25–38Google Scholar
  56. Zanoni M et al (2016) 3D tumor spheroid models for in vitro therapeutic screening: a systematic approach to enhance the biological relevance of data obtained. Sci Rep 6:1–11Google Scholar
  57. Zhang J et al (2015) TGF- b – induced epithelial-to-mesenchymal transition proceeds through stepwise activation of multiple feedback loops. Sci Signal 7:1–12Google Scholar
  58. Zhou Y et al (2013) Multiparameter analyses of three-dimensionally cultured tumor spheroids based on respiratory activity and comprehensive gene expression profiles. Anal Biochem 439:187–193Google Scholar
  59. Zhu J, Xiong G, Trinkle C (2014) Xu, R. integrated extracellular matrix signaling in mammary gland development and breast cancer progression. Histol Histopathol 29:1083–1092PubMedCentralGoogle Scholar
  60. Ziółkowska K et al (2012) Development of a three-dimensional microfluidic system for long-term tumor spheroid culture. Sensors Actuators B Chem 173:908–913Google Scholar

Copyright information

© The International CCN Society 2018

Authors and Affiliations

  1. 1.Department of PathologyUniversity of Michigan Medical SchoolAnn ArborUSA
  2. 2.Molecular and Cellular Pathology Training ProgramUniversity of MichiganAnn ArborUSA
  3. 3.Rogel Cancer CenterUniversity of MichiganAnn ArborUSA
  4. 4.Department of Biomedical Engineering, Biointerfaces InstituteUniversity of MichiganAnn ArborUSA
  5. 5.Wallace H. Coulter Department of Biomedical EngineeringGeorgia Institute of TechnologyAtlantaUSA

Personalised recommendations