Why develop 3D models of tumor cell culture?
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Cell-based assays are usually performed using cells grown on flat surfaces as 2D monolayer cultures. However, only 5% of agents that show in vitro anticancer activity are licensed after demonstrating sufficient efficacy in phase III testing [1]. In the search for more predictive cell-based models, 3D growth cultures have been introduced for drug studies to improve the correlation between cell cultures and tumors [2].
What are 3D Multicellular Tumor Spheroids (MCTS)?Many options for 3D models emerged, taking advantage of both natural and synthetic biomaterials, each with advantages and limitations [3]. Homotypic Multicellular Tumor Spheroids (MCTS) are micro-sized cellular aggregates, comprised exclusively of cancer cells, grown on synthetic biomaterials that prevent cell adhesion to the substrate while maintaining high cell viability [4, 5]. Cells able to secrete endogenous extracellular matrix (ECM) proteins, or with anchorage independent-growth properties, form 3D structures. Some examples of MCTS are shown in Figure 1. We established more than 20 MCTS models for cytotoxicity assays and drug screenings. Check out our Kinetics of MCTS Growth and Survival Assay.
Are the MCTS representative of the in vivo situation?MCTS are able to mimic various features of tumors [6]. The internal structure of MCTS comprises different cell layers, as in solid tumors. The external layer is composed of cells displaying high proliferation rates, while the middle layer is formed of senescent cells and the core contains necrotic cells, consequence of lack of oxygen and nutrients. All cells grow in close contact, reproducing the cell:cell interactions and signaling pathways observed in vivo. Furthermore, various reports point out that gene expression in MCTS is comparable to that observed in tumors [6]. As a result of a tissue-like architecture, some MCTS models have lower sensitivity to anticancer drugs than the same cells in monolayer culture [5, 7-14].
Are there any other advantages of MCTS?An additional advantage of MCTS over monolayer cell culture is in prolonged drug exposure studies. The cells proliferate mainly at the periphery of the spheroid and growth is relatively slow compared with monolayer cultures. Consequently, drug exposures of up to 10 days are possible with high-quality results [5].
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Figure 1. Homotypic MCTS from established cell lines from multiple tumor types.
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1. Hutchinson, L., & Kirk, R. (2011). High drug attrition rates—where are we going wrong? Nature Publishing Group, 8(4), 189–190.
2. Breslin, S., & O’Driscoll, L. (2013). Three-dimensional cell culture: the missing link in drug discovery. Drug Discovery Today, 18(5-6), 240–249.
3. Horvath, P., Aulner, N., Bickle, M., Davies, A. M., Nery, E. D., Ebner, D., et al. (2016). Screening out irrelevant cell-based models of disease. Nature Reviews. Drug Discovery, 15(11), 751–769.
4. Vinci, M., Gowan, S., Boxall, F., Patterson, L., Zimmermann, M., Court, W., et al. (2012). Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biology, 10(1), 29.
5. Selby, M., Delosh, R., Laudeman, J., Ogle, C., Reinhart, R., Silvers, T., et al. (2017). 3D Models of the NCI60 Cell Lines for Screening Oncology Compounds. SLAS Discovery, 22(5), 473–483.
6. Costa, E. C., Moreira, A. F., de Melo-Diogo, D., Gaspar, V. M., Carvalho, M. P., & Correia, I. J. (2016). 3D tumor spheroids: an overview on the tools and techniques used for their analysis. Biotechnology Advances, 34(8), 1427–1441.
7. Sutherland, R. M., Eddy, H. A., Bareham, B., Reich, K., & Vanantwerp, D. (1979). Resistance to adriamycin in multicellular spheroids. International Journal of Radiation Oncology*Biology*Physics, 5(8), 1225–1230.
8. Erlanson, M., Daniel-Szolgay, E., & Carlsson, J. (1992). Relations between the penetration, binding and average concentration of cytostatic drugs in human tumour spheroids. Cancer Chemotherapy and Pharmacology, 29(5), 343–353.
9. Francia, G., Man, S., Teicher, B., Grasso, L., & Kerbel, R. S. (2004). Gene expression analysis of tumor spheroids reveals a role for suppressed DNA mismatch repair in multicellular resistance to alkylating agents. Molecular and Cellular Biology, 24(15), 6837–6849.
10. Hehlgans, S., Eke, I., Storch, K., Haase, M., Baretton, G. B., & Cordes, N. (2009). Caveolin-1 mediated radioresistance of 3D grown pancreatic cancer cells. Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology, 92(3), 362–370.
11. Hirschhaeuser, F., Menne, H., Dittfeld, C., West, J., Mueller-Klieser, W., & Kunz-Schughart, L. A. (2010). Multicellular tumor spheroids: An underestimated tool is catching up again. Journal of Biotechnology, 148(1), 3–15.
12. Karlsson, H., Fryknäs, M., Larsson, R., & Nygren, P. (2012). Loss of cancer drug activity in colon cancer HCT-116 cells during spheroid formation in a new 3-D spheroid cell culture system. Experimental Cell Research, 318(13), 1577–1585.
13. Imamura, Y., Mukohara, T., Shimono, Y., Funakoshi, Y., Chayahara, N., Toyoda, M., et al. (2015). Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. Oncology Reports, 33(4), 1837–1843.
14. Bingel, C., Koeneke, E., Ridinger, J., Bittmann, A., Sill, M., Peterziel, H., et al. (2017). Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance. Cell Death & Disease, 8(8), e3013.
2. Breslin, S., & O’Driscoll, L. (2013). Three-dimensional cell culture: the missing link in drug discovery. Drug Discovery Today, 18(5-6), 240–249.
3. Horvath, P., Aulner, N., Bickle, M., Davies, A. M., Nery, E. D., Ebner, D., et al. (2016). Screening out irrelevant cell-based models of disease. Nature Reviews. Drug Discovery, 15(11), 751–769.
4. Vinci, M., Gowan, S., Boxall, F., Patterson, L., Zimmermann, M., Court, W., et al. (2012). Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation. BMC Biology, 10(1), 29.
5. Selby, M., Delosh, R., Laudeman, J., Ogle, C., Reinhart, R., Silvers, T., et al. (2017). 3D Models of the NCI60 Cell Lines for Screening Oncology Compounds. SLAS Discovery, 22(5), 473–483.
6. Costa, E. C., Moreira, A. F., de Melo-Diogo, D., Gaspar, V. M., Carvalho, M. P., & Correia, I. J. (2016). 3D tumor spheroids: an overview on the tools and techniques used for their analysis. Biotechnology Advances, 34(8), 1427–1441.
7. Sutherland, R. M., Eddy, H. A., Bareham, B., Reich, K., & Vanantwerp, D. (1979). Resistance to adriamycin in multicellular spheroids. International Journal of Radiation Oncology*Biology*Physics, 5(8), 1225–1230.
8. Erlanson, M., Daniel-Szolgay, E., & Carlsson, J. (1992). Relations between the penetration, binding and average concentration of cytostatic drugs in human tumour spheroids. Cancer Chemotherapy and Pharmacology, 29(5), 343–353.
9. Francia, G., Man, S., Teicher, B., Grasso, L., & Kerbel, R. S. (2004). Gene expression analysis of tumor spheroids reveals a role for suppressed DNA mismatch repair in multicellular resistance to alkylating agents. Molecular and Cellular Biology, 24(15), 6837–6849.
10. Hehlgans, S., Eke, I., Storch, K., Haase, M., Baretton, G. B., & Cordes, N. (2009). Caveolin-1 mediated radioresistance of 3D grown pancreatic cancer cells. Radiotherapy and Oncology : Journal of the European Society for Therapeutic Radiology and Oncology, 92(3), 362–370.
11. Hirschhaeuser, F., Menne, H., Dittfeld, C., West, J., Mueller-Klieser, W., & Kunz-Schughart, L. A. (2010). Multicellular tumor spheroids: An underestimated tool is catching up again. Journal of Biotechnology, 148(1), 3–15.
12. Karlsson, H., Fryknäs, M., Larsson, R., & Nygren, P. (2012). Loss of cancer drug activity in colon cancer HCT-116 cells during spheroid formation in a new 3-D spheroid cell culture system. Experimental Cell Research, 318(13), 1577–1585.
13. Imamura, Y., Mukohara, T., Shimono, Y., Funakoshi, Y., Chayahara, N., Toyoda, M., et al. (2015). Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. Oncology Reports, 33(4), 1837–1843.
14. Bingel, C., Koeneke, E., Ridinger, J., Bittmann, A., Sill, M., Peterziel, H., et al. (2017). Three-dimensional tumor cell growth stimulates autophagic flux and recapitulates chemotherapy resistance. Cell Death & Disease, 8(8), e3013.
