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2D and 3D In Vitro Co-Culture for Cancer and Bone Cell Interaction Studies

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Bone Research Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1914))

Abstract

Co-culture assays are used to study the mutual interaction between cells in vitro. This chapter describes 2D and 3D co-culture systems used to study cell-cell signaling crosstalk between cancer cells and bone marrow adipocytes, osteoblasts, osteoclasts, and osteocytes. The chapter provides a step-by-step guide to the most commonly used cell culture techniques, functional assays, and gene expression.

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References

  1. Suva LJ et al (2011) Bone metastasis: mechanisms and therapeutic opportunities. Nat Rev Endocrinol 7(4):208–218

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Waning DL, Guise TA (2014) Molecular mechanisms of bone metastasis and associated muscle weakness. Clin Cancer Res 20(12):3071–3077

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Mundy GR (2002) Metastasis to bone: causes, consequences and therapeutic opportunities. Nat Rev Cancer 2(8):584–593

    Article  CAS  PubMed  Google Scholar 

  4. Roodman GD (2004) Mechanisms of bone metastasis. N Engl J Med 350(16):1655–1664

    Article  CAS  PubMed  Google Scholar 

  5. Croucher PI, McDonald MM, Martin TJ (2016) Bone metastasis: the importance of the neighbourhood. Nat Rev Cancer 16(6):373–386

    Article  CAS  PubMed  Google Scholar 

  6. Kaemmerer E et al (2017) Innovative in vitro models for breast cancer drug discovery. Drug Discov Today Dis Model 21:11–16

    Article  Google Scholar 

  7. Arrigoni C et al (2014) Direct but not indirect co-culture with osteogenically differentiated human bone marrow stromal cells increases RANKL/OPG ratio in human breast cancer cells generating bone metastases. Mol Cancer 13:238

    Article  PubMed  PubMed Central  Google Scholar 

  8. Arrigoni C et al (2016) In vitro co-culture models of breast cancer metastatic progression towards bone. Int J Mol Sci 17(9)

    Article  PubMed Central  Google Scholar 

  9. Sebastian A et al (2015) Cancer-osteoblast interaction reduces sost expression in osteoblasts and up-regulates lncRNA MALAT1 in prostate cancer. Microarrays (Basel) 4(4):503–519

    Article  CAS  Google Scholar 

  10. Chen Y et al (2011) Regulation of breast cancer-induced bone lesions by beta-catenin protein signaling. J Biol Chem 286(49):42575–42584

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Zheng Y et al (2014) Direct crosstalk between cancer and osteoblast lineage cells fuels metastatic growth in bone via auto-amplification of IL-6 and RANKL signaling pathways. J Bone Miner Res 29(9):1938–1949

    Article  CAS  PubMed  Google Scholar 

  12. Nicolin V et al (2008) Breast adenocarcinoma MCF-7 cell line induces spontaneous osteoclastogenesis via a RANK-ligand-dependent pathway. Acta Histochem 110(5):388–396

    Article  CAS  PubMed  Google Scholar 

  13. Bersini S et al (2014) A microfluidic 3D in vitro model for specificity of breast cancer metastasis to bone. Biomaterials 35(8):2454–2461

    Article  CAS  PubMed  Google Scholar 

  14. Di Maggio N et al (2011) Toward modeling the bone marrow niche using scaffold-based 3D culture systems. Biomaterials 32(2):321–329

    Article  PubMed  Google Scholar 

  15. Wang R et al (2005) Three-dimensional co-culture models to study prostate cancer growth, progression, and metastasis to bone. Semin Cancer Biol 15(5):353–364

    Article  CAS  PubMed  Google Scholar 

  16. Orriss IR, Taylor SE, Arnett TR (2012) Rat osteoblast cultures. Methods Mol Biol 816:31–41

    Article  CAS  PubMed  Google Scholar 

  17. Taylor SE, Shah M, Orriss IR (2014) Generation of rodent and human osteoblasts. Bonekey Rep 3:585

    Article  PubMed  PubMed Central  Google Scholar 

  18. Bakker AD, Klein-Nulend J (2012) Osteoblast isolation from murine calvaria and long bones. Methods Mol Biol 816:19–29

    Article  CAS  PubMed  Google Scholar 

  19. Czekanska EM et al (2012) In search of an osteoblast cell model for in vitro research. Eur Cell Mater 24:1–17

    Article  CAS  PubMed  Google Scholar 

  20. Czekanska EM et al (2014) A phenotypic comparison of osteoblast cell lines versus human primary osteoblasts for biomaterials testing. J Biomed Mater Res A 102(8):2636–2643

    Article  CAS  PubMed  Google Scholar 

  21. Costa-Rodrigues J, Fernandes A, Fernandes MH (2011) Reciprocal osteoblastic and osteoclastic modulation in co-cultured MG63 osteosarcoma cells and human osteoclast precursors. J Cell Biochem 112(12):3704–3713

    Article  CAS  PubMed  Google Scholar 

  22. Henriksen K et al (2012) Generation of human osteoclasts from peripheral blood. Methods Mol Biol 816:159–175

    Article  CAS  PubMed  Google Scholar 

  23. Marino S et al (2014) Generation and culture of osteoclasts. Bonekey Rep 3:570

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Abbott RD et al (2016) The use of silk as a scaffold for mature, sustainable unilocular adipose 3D tissue engineered systems. Adv Healthc Mater 5(13):1667–1677

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Moreau JE et al (2007) Tissue-engineered bone serves as a target for metastasis of human breast cancer in a mouse model. Cancer Res 67(21):10304–10308

    Article  CAS  PubMed  Google Scholar 

  26. Kapoor S, Kundu SC (2016) Silk protein-based hydrogels: Promising advanced materials for biomedical applications. Acta Biomater 31:17–32

    Article  CAS  PubMed  Google Scholar 

  27. Zhang X, Reagan MR, Kaplan DL (2009) Electrospun silk biomaterial scaffolds for regenerative medicine. Adv Drug Deliv Rev 61(12):988–1006

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Pallotta I et al (2011) Three-dimensional system for the in vitro study of megakaryocytes and functional platelet production using silk-based vascular tubes. Tissue Eng Part C Methods 17(12):1223–1232

    Article  PubMed  PubMed Central  Google Scholar 

  29. Numata K et al (2011) Spider silk-based gene carriers for tumor cell-specific delivery. Bioconjug Chem 22(8):1605–1610

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Mandal BB, Kundu SC (2009) Non-mulberry silk gland fibroin protein 3-D scaffold for enhanced differentiation of human mesenchymal stem cells into osteocytes. Acta Biomater 5(7):2579–2590

    Article  CAS  PubMed  Google Scholar 

  31. Bellas E et al (2012) In vitro 3D full-thickness skin-equivalent tissue model using silk and collagen biomaterials. Macromol Biosci 12(12):1627–1636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bellas E, Marra KG, Kaplan DL (2013) Sustainable three-dimensional tissue model of human adipose tissue. Tissue Eng Part C Methods 19(10):745–754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Sun L, Reagan MR, Kaplan DL (2010) Role of Cartilage Forming Cells in Regenerative Medicine for Cartilage Repair. Orthop Res Rev 2010(2):85–94

    PubMed  Google Scholar 

  34. Sundelacruz S, Kaplan DL (2009) Stem cell- and scaffold-based tissue engineering approaches to osteochondral regenerative medicine. Semin Cell Dev Biol 20(6):646–655

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Kim HJ et al (2007) Bone regeneration on macroporous aqueous-derived silk 3-D scaffolds. Macromol Biosci 7(5):643–655

    Article  CAS  PubMed  Google Scholar 

  36. Mandal BB et al (2012) High-strength silk protein scaffolds for bone repair. Proc Natl Acad Sci U S A 109(20):7699–7704

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Reagan MR et al (2014) Investigating osteogenic differentiation in multiple myeloma using a novel 3D bone marrow niche model. Blood 124(22):3250–3259

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Correia C et al (2012) Development of silk-based scaffolds for tissue engineering of bone from human adipose-derived stem cells. Acta Biomater 8(7):2483–2492

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Goldstein RH et al (2010) Human bone marrow-derived MSCs can home to orthotopic breast cancer tumors and promote bone metastasis. Cancer Res 70(24):10044–10050

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kwon H et al (2010) Development of an in vitro model to study the impact of BMP-2 on metastasis to bone. J Tissue Eng Regen Med 4(8):590–599

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Momen-Heravi F et al (2013) Current methods for the isolation of extracellular vesicles. Biol Chem 394(10):1253–1262

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Orriss IR et al (2014) Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats. Int J Mol Med 34(5):1201–1208

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Faust J et al (1999) Osteoclast markers accumulate on cells developing from human peripheral blood mononuclear precursors. J Cell Biochem 72(1):67–80

    Article  CAS  PubMed  Google Scholar 

  44. Daigneault M et al (2010) The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages. PLoS One 5(1):e8668

    Article  PubMed  PubMed Central  Google Scholar 

  45. Bodine PV, Vernon SK, Komm BS (1996) Establishment and hormonal regulation of a conditionally transformed preosteocytic cell line from adult human bone. Endocrinology 137(11):4592–4604

    Article  CAS  PubMed  Google Scholar 

  46. Shah KM et al (2016) Osteocyte isolation and culture methods. Bonekey Rep 5:838

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Delgado-Calle J et al (2016) Bidirectional notch signaling and osteocyte-derived factors in the bone marrow microenvironment promote tumor cell proliferation and bone destruction in multiple myeloma. Cancer Res 76(5):1089–1100

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Augst A et al (2008) Effects of chondrogenic and osteogenic regulatory factors on composite constructs grown using human mesenchymal stem cells, silk scaffolds and bioreactors. J R Soc Interface 5(25):929–939

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Wang Y et al (2006) Stem cell-based tissue engineering with silk biomaterials. Biomaterials 27(36):6064–6082

    Article  CAS  PubMed  Google Scholar 

  50. Park SH et al (2010) Relationships between degradability of silk scaffolds and osteogenesis. Biomaterials 31(24):6162–6172

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Kim HJ et al (2008) Bone tissue engineering with premineralized silk scaffolds. Bone 42(6):1226–1234

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We gratefully acknowledge Dr. Aymen I Idris for donating images and his valuable advice and support. S.M. work is supported by the Multiple Myeloma Research Foundation. J.D.C. work is supported by the American Society of Hematology Scholar Award and the International Myeloma Foundation Brian D. Novis Junior Research Grant. M.R.R. the NIH/NIGMS U54GM115516, P30GM106391, P20GM121301, and P30GM103392; the NIH/NIDDK (R24 DK092759-01); the NIH/NIAMS P30AR066261; the American Cancer Society (Research Grant #IRG-16-191-33); and start-up funds from the Maine Medical Center Research Institute.

Author information

Authors and Affiliations

  1. Division Hematology Oncology, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN, USA

    Silvia Marino

  2. Department of Oncology and Metabolism, Medical School, University of Sheffield, Sheffield, UK

    Ryan T. Bishop & Daniëlle de Ridder

  3. Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA

    Jesus Delgado-Calle

  4. Center for Molecular Medicine, Maine Medical Centre Research Institute, Scarborough, ME, USA

    Michaela R. Reagan

Authors
  1. Silvia Marino
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  2. Ryan T. Bishop
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  3. Daniëlle de Ridder
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  4. Jesus Delgado-Calle
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  5. Michaela R. Reagan
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Corresponding author

Correspondence to Silvia Marino .

Editor information

Editors and Affiliations

  1. Department of Oncology and Metabolism, University of Sheffield, Sheffield, UK

    Aymen I. Idris

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Marino, S., Bishop, R.T., de Ridder, D., Delgado-Calle, J., Reagan, M.R. (2019). 2D and 3D In Vitro Co-Culture for Cancer and Bone Cell Interaction Studies. In: Idris, A. (eds) Bone Research Protocols. Methods in Molecular Biology, vol 1914. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8997-3_5

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  • DOI: https://doi.org/10.1007/978-1-4939-8997-3_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8996-6

  • Online ISBN: 978-1-4939-8997-3

  • eBook Packages: Springer Protocols

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