Abstract B65: Live-cell imaging of 3D/4D parallel co-cultures of breast carcinoma cells and breast fibroblasts in tissue architecture and microenvironment engineering (TAME) chambers
Authors: Ji Kyungmin , Zhao Zhiguo , Moin Kamiar , Xu Yong , Sloane Bonnie F. 
Published: May 2016

Interactions among breast carcinoma cells and other cells in the tumor microenvironment, e.g., stromal fibroblasts and immune cells, contribute to malignant progression. We have developed three dimensional (3D) co-culture models and live-cell imaging assays for the analysis of such interactions in real-time, focusing on interactions and associated signaling pathways that might be druggable. Bissell, Brugge and colleagues have elegantly shown that the 3D context of breast cells is essential for their development and neoplastic progression, and the genes down-regulated during acini development in 3D culture are prognostic for clinical outcome of estrogen receptor (ER)-positive and ER-negative breast tumors. We as well as others have demonstrated that 3D cultures of tumor cells can better predict resistance to cytotoxic therapy than 2D monolayer cultures and can be used to identify targets and validate potential therapeutic agents. Nonetheless, in vitro models using only one cell type are at best only partially predictive of in vivo conditions and thus may not be optimal screening platforms for pre-clinical drug discovery. To this end, we have been developing complex 3D co-culture models. We have optimized models that recapitulate in 3D the architecture of the human breast during the transition from normal breast epithelium through pre-malignancy to malignancy and have named them MAME models for mammary architecture and microenvironment engineering models. We have used these models in our laboratory for live-cell imaging and molecular, biochemical and immunochemical analyses in real-time, i.e., 4D (3D + time) of breast tumor cells interacting with other cell types (fibroblasts, myoepithelial cells, lymphatic and blood vessel microvascular endothelial cells, macrophages) in the breast tumor microenvironment. We also developed a live-cell proteolysis assay that allows us to localize and quantify proteolysis and how that is altered by therapeutic approaches and microenvironmental conditions associated with tumors such as acidosis and hypoxia. The MAME models are state-of-the-art in that they allow the analysis of dynamic and temporal processes in live cells and furthermore can be created with all human cells. Thus MAME models provide an alternative to 2D cultures of cancer cells, xenografts of human cancers in mice or transgenic mouse models.

Here we report the development and testing of novel modular chambers designed to allow 3D culture of cancer cells alone or in co-culture (direct and parallel) with other cells over extended periods of time (4D). Because the chambers can be used for cells other than breast, we have designated them tissue architecture and microenvironment engineering (TAME) chambers. With TAME chambers, we have grown for periods ≥60 days human triple negative breast cancer cells, MDA-MB-231, and human breast fibroblasts [normal fibroblasts (NAF98i) and carcinoma-associated fibroblasts (CAF49TKi)]. TAME chambers facilitate collection of conditioned media, without disturbing the cultures, for analyses of dynamic and temporal effects on the secretome; monitoring and quantification by live-cell imaging of dynamic and temporal changes in proliferation, viability, migration, proteolysis and phenotype; regulated introduction of blocking antibodies to cytokines and their receptors; etc. We suggest that TAME chambers will be suitable for high-content imaging and therefore for screening of therapeutic approaches for cancer treatment.

Citation Format: Kyungmin Ji, Zhiguo Zhao, Kamiar Moin, Yong Xu, Bonnie F. Sloane. Live-cell imaging of 3D/4D parallel co-cultures of breast carcinoma cells and breast fibroblasts in tissue architecture and microenvironment engineering (TAME) chambers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B65.