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Engineering a humanized bone organ model in mice to study bone metastases

Nature Protocols volume 12, pages 639–663 (2017)Cite this article

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Abstract

Current in vivo models for investigating human primary bone tumors and cancer metastasis to the bone rely on the injection of human cancer cells into the mouse skeleton. This approach does not mimic species-specific mechanisms occurring in human diseases and may preclude successful clinical translation. We have developed a protocol to engineer humanized bone within immunodeficient hosts, which can be adapted to study the interactions between human cancer cells and a humanized bone microenvironment in vivo. A researcher trained in the principles of tissue engineering will be able to execute the protocol and yield study results within 4–6 months. Additive biomanufactured scaffolds seeded and cultured with human bone-forming cells are implanted ectopically in combination with osteogenic factors into mice to generate a physiological bone 'organ', which is partially humanized. The model comprises human bone cells and secreted extracellular matrix (ECM); however, other components of the engineered tissue, such as the vasculature, are of murine origin. The model can be further humanized through the engraftment of human hematopoietic stem cells (HSCs) that can lead to human hematopoiesis within the murine host. The humanized organ bone model has been well characterized and validated and allows dissection of some of the mechanisms of the bone metastatic processes in prostate and breast cancer.

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Figure 1: Overview of the experimental procedure, with step numbers.
Figure 2: Melt electrospinning machine.
Figure 3: Deposition of the CaP coating on the melt electrospun scaffolds.
Figure 4: Characterization of the human-derived mineralized ECM in vitro.
Figure 5: Characterization of the humanized bone 'organ' in vivo.
Figure 6: Long-term engraftment of human hematopoietic cells following injection of human CD34+ cord blood cells.
Figure 7: Application of the hTEBC to mimic clinical bone metastases.

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References

  1. Coleman, R.E. Clinical features of metastatic bone disease and risk of skeletal morbidity. Clin. Cancer Res. 12, 6243s–6249s (2006).

    Article  PubMed  Google Scholar 

  2. Taubenberger, A.V. In vitro microenvironments to study breast cancer bone colonisation. Adv. Drug Deliv. Rev. 79–80, 135–144 (2014).

    Article  CAS  PubMed  Google Scholar 

  3. Villasante, A., Marturano-Kruik, A. & Vunjak-Novakovic, G. Bioengineered human tumor within a bone niche. Biomaterials 35, 5785–5794 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Tan, P.H., Aung, K.Z., Toh, S.L., Goh, J.C. & Nathan, S.S. Three-dimensional porous silk tumor constructs in the approximation of in vivo osteosarcoma physiology. Biomaterials 32, 6131–6137 (2011).

    Article  CAS  PubMed  Google Scholar 

  5. Hamdi, D.H. et al. In vitro engineering of human 3D chondrosarcoma: a preclinical model relevant for investigations of radiation quality impact. BMC Cancer 15, 579 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Khanna, C. & Hunter, K. Modeling metastasis in vivo. Carcinogenesis 26, 513–523 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Gupta, P.B. & Kuperwasser, C. Disease models of breast cancer. Drug Discov. Today Dis. Models 1, 9–16 (2004).

    Article  CAS  Google Scholar 

  8. Ek, E.T., Dass, C.R. & Choong, P.F. Commonly used mouse models of osteosarcoma. Crit. Rev. Oncol. Hematol. 60, 1–8 (2006).

    Article  PubMed  Google Scholar 

  9. Janeway, K.A. & Walkley, C.R. Modeling human osteosarcoma in the mouse: from bedside to bench. Bone 47, 859–865 (2010).

    Article  PubMed  Google Scholar 

  10. Kretschmann, K.L. & Welm, A.L. Mouse models of breast cancer metastasis to bone. Cancer Metastasis Rev. 31, 579–583 (2012).

    Article  PubMed  Google Scholar 

  11. Singh, A.S. & Figg, W.D. In vivo models of prostate cancer metastasis to bone. J. Urol. 174, 820–826 (2005).

    Article  PubMed  Google Scholar 

  12. Tolcher, A.W. et al. Randomized phase II study of BR96-doxorubicin conjugate in patients with metastatic breast cancer. J. Clin. Oncol. 17, 478–484 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Kostenuik, P.J. et al. Denosumab, a fully human monoclonal antibody to RANKL, inhibits bone resorption and increases BMD in knock-in mice that express chimeric (murine/human) RANKL. J. Bone Miner. Res. 24, 182–195 (2009).

    Article  CAS  PubMed  Google Scholar 

  14. Shtivelman, E. & Namikawa, R. Species-specific metastasis of human tumor cells in the severe combined immunodeficiency mouse engrafted with human tissue. Proc. Natl. Acad. Sci. USA 92, 4661–4665 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Nemeth, J.A. et al. Severe combined immunodeficient-hu model of human prostate cancer metastasis to human bone. Cancer Res. 59, 1987–1993 (1999).

    CAS  PubMed  Google Scholar 

  16. Kuperwasser, C. et al. A mouse model of human breast cancer metastasis to human bone. Cancer Res. 65, 6130–6138 (2005).

    Article  CAS  PubMed  Google Scholar 

  17. Hesami, P. et al. A humanized tissue-engineered in vivo model to dissect interactions between human prostate cancer cells and human bone. Clin. Exp. Metastasis 31, 435–446 (2014).

    Article  CAS  PubMed  Google Scholar 

  18. Holzapfel, B.M. et al. Species-specific homing mechanisms of human prostate cancer metastasis in tissue engineered bone. Biomaterials 35, 4108–4115 (2014).

    Article  CAS  PubMed  Google Scholar 

  19. Thibaudeau, L. et al. A tissue-engineered humanized xenograft model of human breast cancer metastasis to bone. Dis. Model. Mech. 7, 299–309 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Quent, V.M.C., Theodoropoulos, C., Hutmacher, D.W. & Reichert, J.C. Differential osteogenicity of multiple donor-derived human mesenchymal stem cells and osteoblasts in monolayer, scaffold-based 3D culture and in vivo. Biomed. Tech. 61, 253–266 (2016).

    Article  CAS  Google Scholar 

  21. Hutmacher, D.W. Scaffolds in tissue engineering bone and cartilage. Biomaterials 21, 2529–2543 (2000).

    Article  CAS  PubMed  Google Scholar 

  22. Hutmacher, D.W., Schantz, J.T., Lam, C.X., Tan, K.C. & Lim, T.C. State of the art and future directions of scaffold-based bone engineering from a biomaterials perspective. J. Tissue Eng. Regen. Med. 1, 245–260 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Hutmacher, D.W. & Cool, S. Concepts of scaffold-based tissue engineering--the rationale to use solid free-form fabrication techniques. J. Cell. Mol. Med. 11, 654–669 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Reichert, J.C. et al. A tissue engineering solution for segmental defect regeneration in load-bearing long bones. Sci. Transl. Med. 4, 141ra193 (2012).

    Article  CAS  Google Scholar 

  25. Zhou, Y. et al. Combined marrow stromal cell-sheet techniques and high-strength biodegradable composite scaffolds for engineered functional bone grafts. Biomaterials 28, 814–824 (2007).

    Article  CAS  PubMed  Google Scholar 

  26. Brown, T.D. et al. Design and fabrication of tubular scaffolds via direct writing in a melt electrospinning mode. Biointerphases 7, 13 (2012).

    Article  CAS  PubMed  Google Scholar 

  27. Kolambkar, Y.M. et al. Spatiotemporal delivery of bone morphogenetic protein enhances functional repair of segmental bone defects. Bone 49, 485–492 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Hutmacher, D.W. & Dalton, P.D. Melt electrospinning. Chem. Asian J. 6, 44–56 (2011).

    Article  CAS  PubMed  Google Scholar 

  29. Vaquette, C. & Cooper-White, J.J. Increasing electrospun scaffold pore size with tailored collectors for improved cell penetration. Acta Biomater. 7, 2544–2557 (2011).

    Article  CAS  PubMed  Google Scholar 

  30. Brown, T.D., Dalton, P.D. & Hutmacher, D.W. Direct writing by way of melt electrospinning. Adv. Mater. 23, 5651–5657 (2011).

    Article  CAS  PubMed  Google Scholar 

  31. Farrugia, B.L. et al. Dermal fibroblast infiltration of poly(e-caprolactone) scaffolds fabricated by melt electrospinning in a direct writing mode. Biofabrication 5, 025001 (2013).

    Article  CAS  PubMed  Google Scholar 

  32. Vaquette, C., Ivanovski, S., Hamlet, S.M. & Hutmacher, D.W. Effect of culture conditions and calcium phosphate coating on ectopic bone formation. Biomaterials 34, 5538–5551 (2013).

    Article  CAS  PubMed  Google Scholar 

  33. Chatterjea, A., Meijer, G., van Blitterswijk, C. & de Boer, J. Clinical application of human mesenchymal stromal cells for bone tissue engineering. Stem Cells Int. 2010, 215625 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Kern, S., Eichler, H., Stoeve, J., Kluter, H. & Bieback, K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 24, 1294–1301 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Reinisch, A. et al. Epigenetic and in vivo comparison of diverse MSC sources reveals an endochondral signature for human hematopoietic niche formation. Blood 125, 249–260 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Reichert, J.C., Quent, V.M., Noth, U. & Hutmacher, D.W. Ovine cortical osteoblasts outperform bone marrow cells in an ectopic bone assay. J. Tissue Eng. Regen. Med. 5, 831–844 (2011).

    Article  CAS  PubMed  Google Scholar 

  37. Dickhut, A. et al. Calcification or dedifferentiation: requirement to lock mesenchymal stem cells in a desired differentiation stage. J. Cell. Physiol. 219, 219–226 (2009).

    Article  CAS  PubMed  Google Scholar 

  38. Zhang, Z.Y. et al. Superior osteogenic capacity for bone tissue engineering of fetal compared with perinatal and adult mesenchymal stem cells. Stem Cells 27, 126–137 (2009).

    Article  CAS  PubMed  Google Scholar 

  39. Holzapfel, B.M. et al. Tissue engineered humanized bone supports human hematopoiesis in vivo. Biomaterials 61, 103–114 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Ooi, L.L., Zheng, Y., Stalgis-Bilinski, K. & Dunstan, C.R. The bone remodeling environment is a factor in breast cancer bone metastasis. Bone 48, 66–70 (2011).

    Article  PubMed  Google Scholar 

  41. Mastro, A.M., Gay, C.V. & Welch, D.R. The skeleton as a unique environment for breast cancer cells. Clin. Exp. Metastasis 20, 275–284 (2003).

    Article  CAS  PubMed  Google Scholar 

  42. Schneider, J.G., Amend, S.R. & Weilbaecher, K.N. Integrins and bone metastasis: integrating tumor cell and stromal cell interactions. Bone 48, 54–65 (2011).

    Article  CAS  PubMed  Google Scholar 

  43. Patel, L.R., Camacho, D.F., Shiozawa, Y., Pienta, K.J. & Taichman, R.S. Mechanisms of cancer cell metastasis to the bone: a multistep process. Future Oncol. 7, 1285–1297 (2011).

    Article  CAS  PubMed  Google Scholar 

  44. Moreau, J.E. et al. Tissue-engineered bone serves as a target for metastasis of human breast cancer in a mouse model. Cancer Res. 67, 10304–10308 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Taichman, R.S. Blood and bone: two tissues whose fates are intertwined to create the hematopoietic stem-cell niche. Blood 105, 2631–2639 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. Kaplan, R.N. et al. VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche. Nature 438, 820–827 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Shiozawa, Y. et al. Human prostate cancer metastases target the hematopoietic stem cell niche to establish footholds in mouse bone marrow. J. Clin. Invest. 121, 1298–1312 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Sacchetti, B. et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131, 324–336 (2007).

    Article  CAS  PubMed  Google Scholar 

  49. Muerza-Cascante, M.L., Haylock, D., Hutmacher, D.W. & Dalton, P.D. Melt electrospinning and its technologization in tissue engineering. Tissue Eng. Part B Rev. 21, 187–202 (2015).

    Article  CAS  PubMed  Google Scholar 

  50. Song, J. et al. An in vivo model to study and manipulate the hematopoietic stem cell niche. Blood 115, 2592–2600 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Yoshino, H. et al. Natural killer cell depletion by anti-asialo GM1 antiserum treatment enhances human hematopoietic stem cell engraftment in NOD/Shi-scid mice. Bone Marrow Transplant. 26, 1211–1216 (2000).

    Article  CAS  PubMed  Google Scholar 

  52. Ishikawa, F. et al. Development of functional human blood and immune systems in NOD/SCID/IL2 receptor {gamma} chain(null) mice. Blood 106, 1565–1573 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Ito, R., Takahashi, T., Katano, I. & Ito, M. Current advances in humanized mouse models. Cell. Mol. Immunol. 9, 208–214 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Macchiarini, F., Manz, M.G., Palucka, A.K. & Shultz, L.D. Humanized mice: are we there yet? J. Exp. Med. 202, 1307–1311 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lee, J. et al. Implantable microenvironments to attract hematopoietic stem/cancer cells. Proc. Natl. Acad. Sci. USA 109, 19638–19643 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Groen, R.W. et al. Reconstructing the human hematopoietic niche in immunodeficient mice: opportunities for studying primary multiple myeloma. Blood 120, e9–e16 (2012).

    Article  CAS  PubMed  Google Scholar 

  57. Chen, Y. et al. Human extramedullary bone marrow in mice: a novel in vivo model of genetically controlled hematopoietic microenvironment. Blood 119, 4971–4980 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Holzapfel, B.M., Wagner, F., Thibaudeau, L., Levesque, J.P. & Hutmacher, D.W. Concise review: humanized models of tumor immunology in the 21st century: convergence of cancer research and tissue engineering. Stem Cells 33, 1696–1704 (2015).

    Article  CAS  PubMed  Google Scholar 

  59. Thibaudeau, L. et al. Mimicking breast cancer-induced bone metastasis in vivo: current transplantation models and advanced humanized strategies. Cancer Metastasis Rev. 33, 721–735 (2014).

    Article  CAS  PubMed  Google Scholar 

  60. Bersani, F. et al. Bioengineered implantable scaffolds as a tool to study stromal-derived factors in metastatic cancer models. Cancer Res. 74, 7229–7238 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Schuster, J., Zhang, J. & Longo, M. A novel human osteoblast-derived severe combined immunodeficiency mouse model of bone metastasis. J. Neurosurg. Spine 4, 388–391 (2006).

    Article  PubMed  Google Scholar 

  62. Seib, F.P., Berry, J.E., Shiozawa, Y., Taichman, R.S. & Kaplan, D.L. Tissue engineering a surrogate niche for metastatic cancer cells. Biomaterials 51, 313–319 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Goldstein, R.H., Reagan, M.R., Anderson, K., Kaplan, D.L. & Rosenblatt, M. Human bone marrow-derived MSCs can home to orthotopic breast cancer tumors and promote bone metastasis. Cancer Res. 70, 10044–10050 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Battula, V.L. et al. Connective tissue growth factor regulates adipocyte differentiation of mesenchymal stromal cells and facilitates leukemia bone marrow engraftment. Blood 122, 357–366 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Azarin, S.M. et al. In vivo capture and label-free detection of early metastatic cells. Nat. Commun. 6, 8094 (2015).

    Article  PubMed  Google Scholar 

  66. Dondossola, E. et al. Abstract 4941: a humanized bone model for preclinical monitoring of prostate cancer lesions by intravital multiphoton microscopy. Cancer Res. 74, 4941 (2014).

    Google Scholar 

  67. Hughes, C.S., Postovit, L.M. & Lajoie, G.A. Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics 10, 1886–1890 (2010).

    Article  CAS  PubMed  Google Scholar 

  68. Vukicevic, S. et al. Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp. Cell Res. 202, 1–8 (1992).

    Article  CAS  PubMed  Google Scholar 

  69. Thibaudeau, L., Holzapfel, B.M. & Hutmacher, D.W. Humanized mice models for primary bone tumor and bone metastasis research. Cell Cycle 14, 2191–2192 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Holzapfel, B.M. et al. Humanised xenograft models of bone metastasis revisited: novel insights into species-specific mechanisms of cancer cell osteotropism. Cancer Metastasis Rev. 32, 129–145 (2013).

    Article  CAS  PubMed  Google Scholar 

  71. Thibaudeau, L. et al. New mechanistic insights of integrin β1 in breast cancer bone colonization. Oncotarget 6, 332–344 (2015).

    Article  PubMed  Google Scholar 

  72. Wagner, F. et al. A validated preclinical animal model for primary bone tumor research. J. Bone Joint Surg. Am. 98, 916–925 (2016).

    Article  PubMed  Google Scholar 

  73. Kuznetsov, S.A. et al. Age-dependent demise of GNAS-mutated skeletal stem cells and 'normalization' of fibrous dysplasia of bone. J. Bone Miner. Res. 23, 1731–1740 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. Morrison, S.J. & Scadden, D.T. The bone marrow niche for haematopoietic stem cells. Nature 505, 327–334 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Heike, Y., Ohira, T., Takahashi, M. & Saijo, N. Long-term human hematopoiesis in SCID-hu mice bearing transplanted fragments of adult bone and bone marrow cells. Blood 86, 524–530 (1995).

    Article  CAS  PubMed  Google Scholar 

  76. Hubin, F. et al. Maintenance of functional human cancellous bone and human hematopoiesis in NOD/SCID mice. Cell Transplant. 13, 823–831 (2004).

    Article  PubMed  Google Scholar 

  77. Miura, Y. et al. Mesenchymal stem cell-organized bone marrow elements: an alternative hematopoietic progenitor resource. Stem Cells 24, 2428–2436 (2006).

    Article  CAS  PubMed  Google Scholar 

  78. Meyer, L.H. & Debatin, K.M. Diversity of human leukemia xenograft mouse models: implications for disease biology. Cancer Res. 71, 7141–7144 (2011).

    Article  CAS  PubMed  Google Scholar 

  79. Manz, M.G. Human-hemato-lymphoid-system mice: opportunities and challenges. Immunity 26, 537–541 (2007).

    Article  CAS  PubMed  Google Scholar 

  80. Wunderlich, M. et al. AML xenograft efficiency is significantly improved in NOD/SCID-IL2RG mice constitutively expressing human SCF, GM-CSF and IL-3. Leukemia 24, 1785–1788 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Reinisch, A. et al. A humanized bone marrow ossicle xenotransplantation model enables improved engraftment of healthy and leukemic human hematopoietic cells. Nat. Med. 22, 812–821 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Reichert, J.C. et al. Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment. Biomaterials 31, 7928–7936 (2010).

    Article  CAS  PubMed  Google Scholar 

  83. Taubenberger, A.V., Quent, V.M., Thibaudeau, L., Clements, J.A. & Hutmacher, D.W. Delineating breast cancer cell interactions with engineered bone microenvironments. J. Bone Miner. Res. 28, 1399–1411 (2013).

    Article  CAS  PubMed  Google Scholar 

  84. Ding, L., Saunders, T.L., Enikolopov, G. & Morrison, S.J. Endothelial and perivascular cells maintain haematopoietic stem cells. Nature 481, 457–462 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Raymaekers, K., Stegen, S., van Gastel, N. & Carmeliet, G. The vasculature: a vessel for bone metastasis. Bonekey Rep. 4, 742 (2015).

    PubMed  PubMed Central  Google Scholar 

  86. Yardeni, T., Eckhaus, M., Morris, H.D., Huizing, M. & Hoogstraten-Miller, S. Retro-orbital injections in mice. Lab Anim. 40, 155–160 (2011).

    Article  Google Scholar 

  87. Scepansky, E., Goldstein, R. & Rosenblatt, M. Preclinical orthotopic and intracardiac injection models of human breast cancer metastasis to bone and their use in drug discovery. Curr. Protoc. Pharmacol. 52, 14.18 (2011).

    Google Scholar 

  88. Park, S.I., Kim, S.J., McCauley, L.K. & Gallick, G.E. Pre-clinical mouse models of human prostate cancer and their utility in drug discovery. Curr. Protoc. Pharmacol. 51, 14.15 (2010).

    Google Scholar 

  89. Campbell, J.P., Merkel, A.R., Masood-Campbell, S.K., Elefteriou, F. & Sterling, J.A. Models of bone metastasis. J. Vis. Exp (67), e4260 (2012).

    Google Scholar 

  90. Barbier, V., Winkler, I.G. & Levesque, J.P. Mobilization of hematopoietic stem cells by depleting bone marrow macrophages. Methods Mol. Biol. 904, 117–138 (2012).

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the Australian Research Council (Future Fellowship awarded to D.W.H.), the National Health and Medical Research Council (NHMRC Research Fellowship 1044091 to J.-P.L.; NHMRC Project Grant 1082313 to B.M.H., J.-P.L. and D.W.H.), the German Research Foundation (DFG HO 5068/1-1 to B.M.H., B.M.H. and DFG WA 3606/1-1 to F.W.), the National Breast Cancer Foundation (NBCF IN-15-047 to B.M.H. and D.W.H.) and a grant from Worldwide Cancer Research (WWCR 15-11563 to D.W.H.). We thank O. Ramuz, C. Theodoropoulos and J. Baldwin for their help and scientific input. Fibrin glue (TISSEEL Fibrin Sealant) was kindly provided by Baxter Healthcare International. The fibronectin (HFN 7.1) and osteonectin (AON-1) antibodies, developed by R.J. Klebe and J.D. Termine, respectively, were obtained from the Developmental Studies Hybridoma Bank, created by the National Institute of Child Health and Human Development of the National Institutes of Health, and were maintained at the University of Iowa Department of Biology.

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Author notes

  1. Laure C Martine and Boris M Holzapfel: These authors contributed equally to this work.

Authors and Affiliations

  1. Queensland University of Technology (QUT), Brisbane, Queensland, Australia

    Laure C Martine, Boris M Holzapfel, Jacqui A McGovern, Ferdinand Wagner, Verena M Quent, Parisa Hesami, Felix M Wunner, Cedryck Vaquette, Elena M De-Juan-Pardo, Toby D Brown, Dan Jing Wu, Cosmo Orlando Hutmacher, Paul D Dalton, Anna V Taubenberger & Dietmar W Hutmacher

  2. Orthopedic Center for Musculoskeletal Research, University of Wuerzburg, Wuerzburg, Germany

    Boris M Holzapfel

  3. Department of Orthopedics for the University of Regensburg, Asklepios Klinikum Bad Abbach, Bad Abbach, Germany

    Ferdinand Wagner

  4. Department of Pediatric Surgery, Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University of Munich, Munich, Germany

    Ferdinand Wagner

  5. Department of Obstetrics and Gynecology, Martin-Luther-Krankenhaus, Academic Teaching Hospital of the Charité Berlin, Berlin, Germany

    Verena M Quent

  6. The University of Queensland Diamantina Institute, Translational Research Institute, Brisbane, Queensland, Australia

    Bianca Nowlan, Davide Moi & Roberta Mazzieri

  7. Department of Biomedical Engineering, Institute for Complex Molecular Systems, Eindhoven University of Technology, Eindhoven, the Netherlands

    Dan Jing Wu

  8. Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Ontario, Canada

    Tatiana Oussenko, Elia Piccinini & Peter W Zandstra

  9. Stem Cell Biology Group – Blood and Bone Diseases Program, Mater Research Institute – The University of Queensland, Translational Research Institute, Brisbane, Queensland, Australia

    Jean-Pierre Lévesque

  10. Faculty of Medicine and Biomedical Sciences, The University of Queensland, Brisbane, Queensland, Australia

    Jean-Pierre Lévesque

  11. Department of Functional Materials in Medicine and Dentistry, and Bavarian Polymer Institute, University of Wuerzburg, Wuerzburg, Germany

    Paul D Dalton

  12. Biotec TU Dresden, Dresden, Germany

    Anna V Taubenberger

  13. George W Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA

    Dietmar W Hutmacher

  14. Institute for Advanced Study, Technical University Munich, Garching, Germany

    Dietmar W Hutmacher

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  1. Laure C Martine
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  2. Boris M Holzapfel
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  3. Jacqui A McGovern
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  4. Ferdinand Wagner
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  5. Verena M Quent
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  6. Parisa Hesami
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  7. Felix M Wunner
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  9. Elena M De-Juan-Pardo
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  10. Toby D Brown
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  11. Bianca Nowlan
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  13. Cosmo Orlando Hutmacher
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  14. Davide Moi
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  19. Jean-Pierre Lévesque
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  20. Paul D Dalton
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  21. Anna V Taubenberger
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  22. Dietmar W Hutmacher
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L.C.M., B.M.H., A.V.T., J.-P.L., P.Z., R.M. and D.W.H. conceived and designed the experiments. L.C.M., B.M.H., F.W., D.M., D.J.W., C.O.H., T.O., E.P., J.A.M. and B.N. performed the experiments and analyzed the data. L.C.M. wrote the manuscript with the assistance of B.M.H., C.V., E.M.D.-J.-P., F.M.W., J.-P.L., J.A.M. and R.M. C.V. established the CaP coating procedure. T.D.B., P.D.D. and D.W.H. designed and built the custom-made melt electrospinning machine. E.M.D.-J.-P., D.W.H. and F.M.W. developed the scaffold design and fabrication. V.M.Q., P.H. and A.V.T. conducted the first studies to establish the hTEBC model. D.W.H., A.V.T., B.M.H. and L.C.M. supervised the project. All authors read and critiqued the manuscript extensively.

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Correspondence to Dietmar W Hutmacher.

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Integrated supplementary information

Supplementary Figure 1 Long term engraftment and multi-lineage reconstitution in mouse PB.

Flow cytometry gating strategy for hCD45+, hCD34+, CD19+, CD3+ and CD33+ cells. Cells were gated on living cells before cell doublets were excluded. Data was gated on all hCD45+ and mCD45+ cells were to account for hematopoietic lineages before sub-lineage populations were determined. Mouse peripheral blood (PB) and fluorescence minus one (FMO) controls from mice injected with 10,000 CD34+ cord blood cells (16 weeks) and implanted with hTEBC were used as examples of the flow cytometry gating strategy.

Supplementary Figure 2 Long term engraftment and multi-lineage reconstitution in mouse spleen.

Flow cytometry gating strategy for hCD45+, hCD34+, CD19+, CD3+ and CD33+ cells. Cells were gated on living cells before cell doublets were excluded. Data was gated on all hCD45+ and mCD45+ cells were to account for hematopoietic lineages before sub-lineage populations were determined. Cells isolated from mouse spleens and fluorescence minus one (FMO) controls from mice injected with 10,000 hCD34+ cord blood cells (16 weeks) and implanted with hTEBC were used as examples of the flow cytometry gating strategy.

Supplementary Figure 3 Long term engraftment and multi-lineage reconstitution in mouse femur.

Flow cytometry gating strategy for hCD45+, hCD34+, CD19+, CD3+ and CD33+ cells. Cells were gated on living cells before cell doublets were excluded. Data was gated on all hCD45+ and mCD45+ cells were to account for hematopoietic lineages before sub-lineage populations were determined. Analysis of bone marrow from mouse femurs (mBone) and fluorescence minus one (FMO) controls from mice injected with 10,000 hCD34+ cord blood cells (16 weeks) and implanted with hTEBC were used as examples of the flow cytometry gating strategy.

Supplementary Figure 4 Long term engraftment and multi-lineage reconstitution in hTEBC

Flow cytometry gating strategy for hCD45+, hCD34+, CD19+, CD3+ and CD33+ cells. Cells were gated on living cells before cell doublets were excluded. Data was gated on all hCD45+ and mCD45+ cells were to account for hematopoietic lineages before sub-lineage populations were determined. Analysis of bone marrow from the hTEBC and fluorescence minus one (FMO) controls from mice injected with 10,000 hCD34+ cord blood cells (16 weeks) and implanted with hTEBC were used as examples of the flow cytometry gating strategy. Note that the FMO controls are the same as used in Supplementary Figure 3.

Supplementary Figure 5 Multi-lineage reconstitution in mice peripheral blood and organs following hCD34+ cell engraftment

Mice were engrafted with either (a, c) 10,000 or (b, d) 50,000 hCD34+ cord blood cells. Flow cytometry analysis was performed and gated on the total population of mCD45+ and hCD45+ cells to detect (a, b) the time course of the relative hCD19+, hCD3+ and hCD33+ cell engraftment in mouse PB. (c, d) The organ distribution and engraftment of hCD19+, hCD3+ and hCD33+ cells within the total population of mCD45+ and hCD45+ cells at the experimental endpoint (16-24 weeks). Data is represented as mean ± s.e.m, n=3 for mice without a hTEBC and n=4 for mice with a hTEBC.

Supplementary Figure 6 Engraftment and multi-lineage reconstitution of mouse femur and hTEBC.

Flow cytometry gating strategy for hCD45+, hCD34+, hCD38+, hCD3+, hCD19+ and CD11b+ following engraftment with adult bone-derived hCD34+ cells. Analysis of bone marrow isolated from (a) mouse femur and (b) hTEBC from mice 5 weeks after injection with 2 × 105 hCD34+ cells.

Supplementary Figure 7 Controls for species-specificity of antibodies used for immunohistochemical analysis.

Human and mouse tissues were used to serve respectively as positive and negative controls to test the human-specificity of antibodies against human NuMA, lamin A/C, mitochondria, collagen type I, osteocalcin, CD146, CD45 and CD34. Details of antigen retrieval methods, antibody suppliers and antibody dilutions can be found in Supplementary Table 1.

Supplementary information

Supplementary Figures and Tables

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1854 kb)

Melt electrospinning of tubular mPCL scaffolds. (MOV 12856 kb)

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Martine, L., Holzapfel, B., McGovern, J. et al. Engineering a humanized bone organ model in mice to study bone metastases. Nat Protoc 12, 639–663 (2017). https://doi.org/10.1038/nprot.2017.002

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