time of assay, the data relate to changes in processes governed by cell cycle or cell death. As it is, decreasing the proliferation rate is generally correlated negatively to cell health where there is either a decrease in cell cycle progression or an increase in cell death. Likewise, dramatic increases in cell proliferation rates may be associated with loss of cell cycle control and an increase in disease pathogenesis.
The adaptation of 3D culture for studying cellular proliferation demonstrates that the ECM may impact the pharmacological dose response, and by evaluating cells in the proper context, these models elucidate the potential for drug resistance and accelerate translational research. Neoplastic cells in 3D culture have demonstrated drug resistance to many chemotherapeutic agents including doxorubicin, etoposide, cisplatin, and the nitrogen mustard, (10) and these cultures have been shown to promote resistance to ionizing and ultraviolet radiation as well. (11, 12)
Since cell–matrix interactions are mediated via cell surface receptors, know as integrins, and their subsequent signaling cascades, the search for targets that can abrogate the protective effects of the ECM and sensitize cancers to existing treatment therapies has begun, and these assays will be instrumental in identifying these targets and screening therapeutic compounds. During this process, stripwells from a 96-well plate are coated with ECM proteins which form hydrogels that mimic the tissue microenvironment. When cells are added to these wells, specific interactions between the cell and the ECM proteins induce a change in the cells’ biological program, and as the cells proliferate, they assume structural and functional characteristics as seen in vivo. Figure 1 demonstrates differences in cell morphology and structure formation for normal, MCF-10A, and malignant, MDA-MB-231, mammary epithelial cells cultured in 2D and 3D cultures. The MCF-10A cells grow in discrete cobblestone monolayers on plastic, and form glandular structures when cultured on BME; likewise, the MDA-MB-231 cells grow in spindle-shaped monolayers on plastic and form disorganized masses when cultured on BME. Cells may be monitored via microscopy during the course of the assay to evaluate cell morphology; structure formation; and, to some extent, viability. At the end of the culture, the 3D culture cell proliferation reagent is added to each well, and the viable cells within the well convert the substrate to a yellow product that absorbs at 450 nm. The absorbance can be evaluated in a 96-well plate reader, and the relative proliferation can be determined based on these absorbance values.
Figure 1. 3D cultures promote cell assembly as seen in vivo. The normal mammary epithelial cell line, MCF-10A, grows as a cobblestone monolayer on plastic (a) and forms glandular structures in a 3D BME culture (b). The malignant mammary epithelial cell line, MDAMB-231, grows as spindle-shaped monolayers on plastic (c) and forms a disorganized mass in a 3D BME culture (d).
References
1. Birgersdotter, A.; Sandberg, R.; Ernberg, I. Gene expression perturbation in vitro—a growing case for three-dimensional (3-D) culture systems. Semin. Cancer Biol. 2005, 15(5), 405–12.
2. Cukierman, E.; Pankov, R.; Yamada, K.M. Cell interactions with three-dimensional matrices. Curr. Opin. Cell Biol. 2002, 14(5), 633–40.
3. Nelson, C.M.; Bissell, M.J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Ann. Rev. Cell Dev. Biol. 2006, 22, 287–309.
4. Schuetz, E.G.; Li, D.; Omiecinski, C.J.; Muller-Eberhard, U.; Kleinman, H.K.; Elswick, B.; Guzelian, P.S. Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix. J. Cell Physiol. 1988, 134(3), 309–23.
5. Barcellos-Hoff, M.H.; Aggeler, J.; Ram, T.G.; Bissell, M.J. Functional differentiation and alveolar morphogenesis of primary mammary cultures on reconstituted basement membrane. Development 1989, 105(2), 223–35.
6. Webber, M.M.; Bello, D.; Kleinman, H.K.; Hoffman, M.P. Acinar differentiation by non-malignant immortalized human prostatic epithelial cells and its loss by malignant cells. Carcinogenesis 1997, 18(6), 1225–31.
7. Grant, D.S.; Tashiro, K.; Segui-Real, B.; Yamada, Y.; Martin, G.R.; Kleinman, H.K. Two different laminin domains mediate the differentiation of human endothelial cells into capillary-like structures in vitro. Cell 1989, 58(5), 933–43.
8. Griffith, L.G.; Swartz, M.A. Capturing complex 3-D tissue physiology in vitro. Nat. Rev. Mol. Cell Biol. 2006, 7(3), 211–24.
9. Petersen, O.W.; Ronnov-Jessen, L.; Howlett, A.R.; Bissell, M.J. Interaction with Basement Membrane Serves to Rapidly Distinguish Growth and Differentiation Pattern of Normal and Malignant Human Breast Epithelial Cells. Proceedings of the National Academy of Sciences 1992, 89(19), 9064–8.
10. Fridman, R.; Giaccone, G.; Kanemoto, T.; Martin, G.R.; Gazdar, A.F.; Mulshine, J.L. Reconstituted Basement Membrane (Matrigel) and Laminin Can Enhance the Tumorigenicity and the Drug Resistance of Small Cell Lung Cancer Cell Lines. Proceedings of the National Academy of Sciences 1990, 87(17), 6698–6702.
11. Hodkinson, P.S.; Elliott, T.; Wong, W.S.; Rintoul, R.C.; Mackinnon, A.C.; Haslett, C.; Sethi, T. ECM overrides DNA damage-induced cell cycle arrest and apoptosis in small-cell lung cancer cells through beta1 integrin-dependent activation of PI3-kinase. Cell Death Differ. 2006, 13(10), 1776–88.
12. Pogany, G.; Timar, F.; Olah, J.; Harisi, R.; Polony, G.; Paku, S.; Bocsi, J.; Jeney, A.; Laurie, G.W. Role of the basement membrane in tumor cell dormancy and cytotoxic resistance. Oncology 2001, 60(3), 274–81.
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