Thesis

Developing 3D spheroid cultures for functional assays of osteotropic breast cancer

Traditional cell culture techniques grow cells in a two-dimensional (2D) format on compatible surfaces, but cells do not grow like this in vivo. A fast-emerging alternative is three-dimensional (3D) cell culture, which encourages cells to work together while suspended in a matrix rather than relying on adhesion to a treated surface. Most studies utilize a commercially available, industry standard recombinant basement membrane substitute--Matrigel ; however, this is proprietary and expensive. For these studies, my goal was to refine an alternative to Matrigel for use as a suspension media and surrogate extra-cellular matrix (ECM). My efforts focused on economical methylcellulose (a synthetic derivative of cellulose dissolved into basal growth media and called Methocel) in combination with rat-tail collagen (Type I). Optimized concentrations were evaluated towards the formation of spheroids among several cell lines including all three isogenic variants of a metastatic breast cancer cell line, MDA-MB-231, notoriously difficult to adapt to spheroid culture. I chose to use the metastatic breast cancer cell line MDA-MB-231 and two isogenic variants, MDA-MB-231Bo and MDA-MB-231Br, shown to regularly metastasize to either bone or brain respectively. This cell line series was used to evaluate the potential of this low-cost matrix to facilitate spheroid cultures; to determine expression changes in the canonical cell adhesion components Integrins (2, β1, and β4), as well as, E-Cadherins (ECAD) and N-Cadherins (NCAD); and to evaluate the functional significance of the spheroids to a relevant drug treatment. Here, by image analysis of spheroid metastatic progression and semi-quantitative RT-PCR analysis of metastatic cancer cells, we show the potential of this system in observing cancer cell migration and cellular behavior under more in vivo-like conditions, bridging the gap between basic two-dimensional cell culture and the animal model. Furthermore, through the use of this powerful model, we showed the efficacy of zoledronic acid to inhibit osteotropic breast cancer cell migration by up to ~14% post-72h treatment time. Lastly, zoledronic acid treated spheroids showed a continued improvement towards regaining more epithelial-like gene transcription profiles in genes associated with cells having undergone epithelial-mesenchyme transition. Together, these data add validity to our claim that metastatic studies using a surrogate extracellular matrix, such the one suggested in this study, continue to bridge the gap between typical two-dimensional cell culture and the animal model.

Relationships

Items