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

  • Sabra I. Djomehri
  • Boris Burman
  • Maria E. Gonzalez
  • Shuichi Takayama
  • Celina G. Kleer
Nuts and Bolts
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Abstract

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.

Keywords

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

Notes

Acknowledgements

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)

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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

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