Skip to main content

Advertisement

SpringerLink
Log in

Spatio-temporal modeling and live-cell imaging of proteolysis in the 4D microenvironment of breast cancer

  • Published:
Cancer and Metastasis Reviews Aims and scope Submit manuscript

Abstract

Cells grown in three dimensions (3D) within natural extracellular matrices or synthetic scaffolds more closely recapitulate the phenotype of those cells within tissues in regard to normal developmental and pathobiological processes. This includes degradation of the surrounding stroma as the cells migrate and invade through the matrices. As 3D cultures of tumor cells predict efficacy of, and resistance to, a wide variety of cancer therapies, we employed tissue-engineering approaches to establish 3D pathomimetic avatars of human breast cancer cells alone and in the context of both their cellular and pathochemical microenvironments. We have shown that we can localize and quantify key parameters of malignant progression by live-cell imaging of the 3D avatars over time (4D). One surrogate for changes in malignant progression is matrix degradation, which can be localized and quantified by our live-cell proteolysis assay. This assay is predictive of changes in spatio-temporal and dynamic interactions among the co-cultured cells and changes in viability, proliferation, and malignant phenotype. Furthermore, our live-cell proteolysis assay measures the effect of small-molecule inhibitors of proteases and kinases, neutralizing or blocking antibodies to cytokines and photodynamic therapy on malignant progression. We suggest that 3D/4D pathomimetic avatars in combination with our live-cell proteolysis assays will be a useful preclinical screening platform for cancer therapies. Our ultimate goal is to develop 3D/4D avatars from an individual patient’s cancer in which we can screen “personalized medicine” therapies using changes in proteolytic activity to quantify therapeutic efficacy.

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

Access this article

Log in via an institution

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

Instant access to the full article PDF.

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

Similar content being viewed by others

References

  1. Debnath, J., & Brugge, J. S. (2005). Modelling glandular epithelial cancers in three-dimensional cultures. Nature Reviews. Cancer, 5(9), 675–688. https://doi.org/10.1038/nrc1695.

    Article  CAS  PubMed  Google Scholar 

  2. Martin, K. J., Patrick, D. R., Bissell, M. J., & Fournier, M. V. (2008). Prognostic breast cancer signature identified from 3D culture model accurately predicts clinical outcome across independent datasets. PLoS One, 3(8), e2994. https://doi.org/10.1371/journal.pone.0002994.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Weigelt, B., Ghajar, C. M., & Bissell, M. J. (2014). The need for complex 3D culture models to unravel novel pathways and identify accurate biomarkers in breast cancer. Advanced Drug Delivery Reviews, 69-70, 42–51. https://doi.org/10.1016/j.addr.2014.01.001.

    Article  CAS  PubMed  Google Scholar 

  4. Siegel, R. L., Miller, K. D., & Jemal, A. (2019). Cancer statistics, 2019. CA: a Cancer Journal for Clinicians, 69(1), 7–34. https://doi.org/10.3322/caac.21551.

    Article  Google Scholar 

  5. Li, Q., Mullins, S. R., Sloane, B. F., & Mattingly, R. R. (2008). p21-activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer. Neoplasia, 10(4), 314–329.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Li, Q., Chow, A. B., & Mattingly, R. R. (2010). Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors. The Journal of Pharmacology and Experimental Therapeutics, 332(3), 821–828. https://doi.org/10.1124/jpet.109.160390.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Nam, J. M., Onodera, Y., Bissell, M. J., & Park, C. C. (2010). Breast cancer cells in three-dimensional culture display an enhanced radioresponse after coordinate targeting of integrin alpha5beta1 and fibronectin. Cancer Research, 70(13), 5238–5248. https://doi.org/10.1158/0008-5472.CAN-09-2319.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Maguire, S. L., Peck, B., Wai, P. T., Campbell, J., Barker, H., Gulati, A., Daley, F., Vyse, S., Huang, P., Lord, C. J., Farnie, G., Brennan, K., & Natrajan, R. (2016). Three-dimensional modelling identifies novel genetic dependencies associated with breast cancer progression in the isogenic MCF10 model. The Journal of Pathology, 240(3), 315–328. https://doi.org/10.1002/path.4778.

    Article  CAS  PubMed  Google Scholar 

  9. Brock, E. J., Ji, K., Shah, S., Mattingly, R. R., & Sloane, B. F. (2019). In vitro models for studying invasive transitions of ductal carcinoma in situ. Journal of Mammary Gland Biology and Neoplasia, 24(1), 1–15. https://doi.org/10.1007/s10911-018-9405-3.

    Article  PubMed  Google Scholar 

  10. Herschkowitz, J. I., & Behbod, F. (2018). Advances in DCIS research and treatment. [journal issue]. Journal of Mammary Gland Biology and Neoplasia, 23 & 24, 1–301. https://doi.org/10.1007/s10911-018-9419-x.

    Article  Google Scholar 

  11. Edwards, D., Hoyer-Hansen, G., Blasi, F., & Sloane, B. F. (2008). The cancer degradome: protease and cancer biology. New York: Springer.

    Book  Google Scholar 

  12. Sloane, B. F., List, K., Fingleton, B., & Matrisian, L. (2013). Proteases: structure and function. New York: Springer.

    Google Scholar 

  13. Darvishian, F., Ozerdem, U., Adams, S., Chun, J., Pirraglia, E., Kaplowitz, E., Guth, A., Axelrod, D., Shapiro, R., Price, A., Troxel, A., Schnabel, F., & Roses, D. (2019). Tumor-infiltrating lymphocytes in a contemporary cohort of women with ductal carcinoma in situ (DCIS). Annals of Surgical Oncology, 26(10), 3337–3343. https://doi.org/10.1245/s10434-019-07562-x.

    Article  PubMed  Google Scholar 

  14. Grugan, K. D., McCabe, F. L., Kinder, M., Greenplate, A. R., Harman, B. C., Ekert, J. E., van Rooijen, N., Anderson, G. M., Nemeth, J. A., Strohl, W. R., Jordan, R. E., & Brezski, R. J. (2012). Tumor-associated macrophages promote invasion while retaining Fc-dependent anti-tumor function. Journal of Immunology, 189(11), 5457–5466. https://doi.org/10.4049/jimmunol.1201889.

    Article  CAS  Google Scholar 

  15. Shree, T., Olson, O. C., Elie, B. T., Kester, J. C., Garfall, A. L., Simpson, K., Bell-McGuinn, K. M., Zabor, E. C., Brogi, E., & Joyce, J. A. (2011). Macrophages and cathepsin proteases blunt chemotherapeutic response in breast cancer. Genes & Development, 25(23), 2465–2479. https://doi.org/10.1101/gad.180331.111.

    Article  CAS  Google Scholar 

  16. Erdogan, B., & Webb, D. J. (2017). Cancer-associated fibroblasts modulate growth factor signaling and extracellular matrix remodeling to regulate tumor metastasis. Biochemical Society Transactions, 45(1), 229–236. https://doi.org/10.1042/BST20160387.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Qiu, S. Q., Waaijer, S. J. H., Zwager, M. C., de Vries, E. G. E., van der Vegt, B., & Schroder, C. P. (2018). Tumor-associated macrophages in breast cancer: Innocent bystander or important player? Cancer Treatment Reviews, 70, 178–189. https://doi.org/10.1016/j.ctrv.2018.08.010.

    Article  CAS  PubMed  Google Scholar 

  18. Dawson, P. J., Wolman, S. R., Tait, L., Heppner, G. H., & Miller, F. R. (1996). MCF10AT: a model for the evolution of cancer from proliferative breast disease. The American Journal of Pathology, 148(1), 313–319.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Santner, S. J., Dawson, P. J., Tait, L., Soule, H. D., Eliason, J., Mohamed, A. N., Wolman, S. R., Heppner, G. H., & Miller, F. R. (2001). Malignant MCF10CA1 cell lines derived from premalignant human breast epithelial MCF10AT cells. Breast Cancer Research and Treatment, 65(2), 101–110.

    Article  CAS  PubMed  Google Scholar 

  20. Miller, F. R., Santner, S. J., Tait, L., & Dawson, P. J. (2000). MCF10DCIS.com xenograft model of human comedo ductal carcinoma in situ. Journal of the National Cancer Institute, 92(14), 1185–1186. https://doi.org/10.1093/jnci/92.14.1185a.

    Article  CAS  PubMed  Google Scholar 

  21. Sameni, M., Cavallo-Medved, D., Franco, O. E., Chalasani, A., Ji, K., Aggarwal, N., Anbalagan, A., Chen, X., Mattingly, R. R., Hayward, S. W., & Sloane, B. F. (2017). Pathomimetic avatars reveal divergent roles of microenvironment in invasive transition of ductal carcinoma in situ. Breast Cancer Research, 19(1), 56. https://doi.org/10.1186/s13058-017-0847-0.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Polyak, K., & Hu, M. (2005). Do myoepithelial cells hold the key for breast tumor progression? Journal of Mammary Gland Biology and Neoplasia, 10(3), 231–247. https://doi.org/10.1007/s10911-005-9584-6.

    Article  PubMed  Google Scholar 

  23. Labernadie, A., Kato, T., Brugues, A., Serra-Picamal, X., Derzsi, S., Arwert, E., et al. (2017). A mechanically active heterotypic E-cadherin/N-cadherin adhesion enables fibroblasts to drive cancer cell invasion. Nature Cell Biology, 19(3), 224–237. https://doi.org/10.1038/ncb3478.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Sameni, M., Tovar, E. A., Essenburg, C. J., Chalasani, A., Linklater, E. S., Borgman, A., Cherba, D. M., Anbalagan, A., Winn, M. E., Graveel, C. R., & Sloane, B. F. (2016). Cabozantinib (XL184) inhibits growth and invasion of preclinical TNBC models. Clinical Cancer Research, 22(4), 923–934. https://doi.org/10.1158/1078-0432.CCR-15-0187.

    Article  CAS  PubMed  Google Scholar 

  25. Cavallo-Medved, D., Rudy, D., Blum, G., Bogyo, M., Caglic, D., & Sloane, B. F. (2009). Live-cell imaging demonstrates extracellular matrix degradation in association with active cathepsin B in caveolae of endothelial cells during tube formation. Experimental Cell Research, 315(7), 1234–1246. https://doi.org/10.1016/j.yexcr.2009.01.021.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Osuala, K. O., Sameni, M., Shah, S., Aggarwal, N., Simonait, M. L., Franco, O. E., Hong, Y., Hayward, S. W., Behbod, F., Mattingly, R. R., & Sloane, B. F. (2015). Il-6 signaling between ductal carcinoma in situ cells and carcinoma-associated fibroblasts mediates tumor cell growth and migration. BMC Cancer, 15, 584. https://doi.org/10.1186/s12885-015-1576-3.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Jedeszko, C., Sameni, M., Olive, M. B., Moin, K., & Sloane, B. F. (2008). Visualizing protease activity in living cells: from two dimensions to four dimensions. Current Protocols in Cell Biology, 39(1), 4.20.1–4.20. https://doi.org/10.1002/0471143030.cb0420s39.

    Article  Google Scholar 

  28. Chalasani, A., Ji, K., Sameni, M., Mazumder, S. H., Xu, Y., Moin, K., et al. (2017). Live-cell imaging of protease activity: assays to screen therapeutic approaches. Methods in Molecular Biology, 1574, 215–225. https://doi.org/10.1007/978-1-4939-6850-3_16.

    Article  CAS  PubMed  Google Scholar 

  29. Ji, K., Mayernik, L., Moin, K., & Sloane, B. F. (2019). Acidosis and proteolysis in the tumor microenvironment. Cancer Metastasis Reviews, 38(1–2), 103–112. https://doi.org/10.1007/s10555-019-09796-3.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Rothberg, J. M., Bailey, K. M., Wojtkowiak, J. W., Ben-Nun, Y., Bogyo, M., Weber, E., Moin, K., Blum, G., Mattingly, R. R., Gillies, R. J., & Sloane, B. F. (2013). Acid-mediated tumor proteolysis: contribution of cysteine cathepsins. Neoplasia, 15(10), 1125–1137. https://doi.org/10.1593/neo.13946.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Curino, A. C., Engelholm, L. H., Yamada, S. S., Holmbeck, K., Lund, L. R., Molinolo, A. A., Behrendt, N., Nielsen, B. S., & Bugge, T. H. (2005). Intracellular collagen degradation mediated by uPARAP/Endo180 is a major pathway of extracellular matrix turnover during malignancy. The Journal of Cell Biology, 169(6), 977–985. https://doi.org/10.1083/jcb.200411153.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Hanahan, D., & Weinberg, R. A. (2011). Hallmarks of cancer: the next generation. Cell, 144(5), 646–674. https://doi.org/10.1016/j.cell.2011.02.013.

    Article  CAS  PubMed  Google Scholar 

  33. Estrella, V., Chen, T., Lloyd, M., Wojtkowiak, J., Cornnell, H. H., Ibrahim-Hashim, A., Bailey, K., Balagurunathan, Y., Rothberg, J. M., Sloane, B. F., Johnson, J., Gatenby, R. A., & Gillies, R. J. (2013). Acidity generated by the tumor microenvironment drives local invasion. Cancer Research, 73(5), 1524–1535. https://doi.org/10.1158/0008-5472.CAN-12-2796.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Edgington, L. E., Verdoes, M., & Bogyo, M. (2011). Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes. Current Opinion in Chemical Biology, 15(6), 798–805. https://doi.org/10.1016/j.cbpa.2011.10.012.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Blum, G., Mullins, S. R., Keren, K., Fonovic, M., Jedeszko, C., Rice, M. J., et al. (2005). Dynamic imaging of protease activity with fluorescently quenched activity-based probes. Nature Chemical Biology, 1(4), 203–209. https://doi.org/10.1038/nchembio728.

    Article  CAS  PubMed  Google Scholar 

  36. Withana, N. P., Blum, G., Sameni, M., Slaney, C., Anbalagan, A., Olive, M. B., Bidwell, B. N., Edgington, L., Wang, L., Moin, K., Sloane, B. F., Anderson, R. L., Bogyo, M. S., & Parker, B. S. (2012). Cathepsin B inhibition limits bone metastasis in breast cancer. Cancer Research, 72(5), 1199–1209. https://doi.org/10.1158/0008-5472.CAN-11-2759.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Duivenvoorden, H. M., Rautela, J., Edgington-Mitchell, L. E., Spurling, A., Greening, D. W., Nowell, C. J., Molloy, T. J., Robbins, E., Brockwell, N. K., Lee, C. S., Chen, M., Holliday, A., Selinger, C. I., Hu, M., Britt, K. L., Stroud, D. A., Bogyo, M., Möller, A., Polyak, K., Sloane, B. F., O'Toole, S. A., & Parker, B. S. (2017). Myoepithelial cell-specific expression of stefin A as a suppressor of early breast cancer invasion. The Journal of Pathology, 243(4), 496–509. https://doi.org/10.1002/path.4990.

    Article  CAS  PubMed  Google Scholar 

  38. Kasperkiewicz, P., Altman, Y., D'Angelo, M., Salvesen, G. S., & Drag, M. (2017). Toolbox of fluorescent probes for parallel imaging reveals uneven location of serine proteases in neutrophils. Journal of the American Chemical Society, 139(29), 10115–10125. https://doi.org/10.1021/jacs.7b04394.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Heuser, J. (1989). Changes in lysosome shape and distribution correlated with changes in cytoplasmic pH. The Journal of Cell Biology, 108(3), 855–864. https://doi.org/10.1083/jcb.108.3.855.

    Article  CAS  PubMed  Google Scholar 

  40. Rozhin, J., Sameni, M., Ziegler, G., & Sloane, B. F. (1994). Pericellular pH affects distribution and secretion of cathepsin B in malignant cells. Cancer Research, 54(24), 6517–6525.

    CAS  PubMed  Google Scholar 

  41. Moffat, J. G., Rudolph, J., & Bailey, D. (2014). Phenotypic screening in cancer drug discovery - past, present and future. Nature Reviews. Drug Discovery, 13(8), 588–602. https://doi.org/10.1038/nrd4366.

    Article  CAS  PubMed  Google Scholar 

  42. Yang, Z. Q., Albertson, D., & Ethier, S. P. (2004). Genomic organization of the 8p11-p12 amplicon in three breast cancer cell lines. Cancer Genetics and Cytogenetics, 155(1), 57–62. https://doi.org/10.1016/j.cancergencyto.2004.03.013.

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This work was supported in part by National Institute of Health grants R01 CA131990 (RRM and BFS) and R21 CA1759331 (BFS), a Department of Defense Breast Cancer Research Program Postdoctoral Fellowship Award (W81XWH-12-1-0024; KO), and an award from the President’s Research Enhancement Program of Wayne State University (BFS). Imaging was performed in the Microscopy, Imaging and Cytometry Resources Core (KM), which is supported, in part, by National Institutes of Health Center grant P30 CA022453 to the Karmanos Cancer Institute at Wayne State University, and the Perinatology Research Branch of the National Institute of Child Health and Development at Wayne State University.

Author information

Authors and Affiliations

  1. Department of Pharmacology, Wayne State University, Detroit, MI, 48201, USA

    Kyungmin Ji, Mansoureh Sameni, Kingsley Osuala, Kamiar Moin, Raymond R. Mattingly & Bonnie F. Sloane

Authors
  1. Kyungmin Ji
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mansoureh Sameni
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kingsley Osuala
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kamiar Moin
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Raymond R. Mattingly
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Bonnie F. Sloane
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Bonnie F. Sloane.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ji, K., Sameni, M., Osuala, K. et al. Spatio-temporal modeling and live-cell imaging of proteolysis in the 4D microenvironment of breast cancer. Cancer Metastasis Rev 38, 445–454 (2019). https://doi.org/10.1007/s10555-019-09810-8

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10555-019-09810-8

Keywords

Search

Navigation