Abstract P3-03-07: High-throughput pharmacogenomic platform to functionally probe breast cancer vulnerabilities in the context of the 3-dimensional tumor microenvironment
Authors: Dhimolea E , De Matos Simoes R , Awate P , Tang H , Culhane A , Mitsiades C 
Published: March 2017

The pharmacological vulnerabilities of malignant cells have been traditionally evaluated by the use of cell lines grown in vitro in 2-dimensional (2D) plastic surfaces. In contrast, malignant tumors in vivo grow as 3-dimensional (3D) cellular masses. We used Estrogen Receptor (ER)-positive (MCF7, ZR75-1 and T47D) and triple-negative (MDA-MB-231) breast cancer (BrCa) cells to assess the differences between the malignant growth in 2D vs. 3D conditions with regards to morphology, molecular characteristics and response to therapeutic agents. The ER-positive BrCa cells formed compact spheroids when grown in collagen type I or matrigel; in contrast the MDA-MB-231 cells assumed an invasive phenotype characterized by infiltrating bundles of cells. Antiestrogens induced acinar differentiation in the compact spheroid morphology of ER-positive BrCa cells. This effect of antiestrogens was blocked in co-cultures of BrCa cells with bone marrow stromal cells, suggesting that the 3-dimensional microenvironment of distant metastatic sites can affect the susceptibility of BrCa cells to anti-tumor agents. We further probed this question through pharmacological screens of ~100 FDA-approved antineoplastic agents and 400 target-annotated kinase inhibitors, to assess the pathophysiological relevance of these in vitro interactions and to identify specific BrCa vulnerabilities in 3D conditions. The 3D spheroid morphology was associated with significant decrease in the efficacy of several classes of conventional DNA-damaging (e.g. anthracyclins) and anti-microtubule (e.g. vinca alkaloids) agents, proteasome inhibitors, as well as recently established targeted therapies, includding kinase inhibitors. This differential 3D spheroid-associated drug resistance vs. sensitization was more pronounced for the triple-negative MDA-MB-231 cell line compared to the ER-positive BrCa cells. Interestingly, inhibition of folate metabolism induced reduced viability of ER-positive BrCa spheroids, indicating increased dependency of ER-positive tumor cells on this pathway, under 3D growth conditions. Interestingly, mut-p53 T47D 3D spheroids were resistant to antifolates, suggesting a possible role of functional p53 for the anti-BrCa effect of antifolates in 3D conditions. Gene expression analysis and reverse-phase protein array analyses of BrCa cells in this system indicated that growth of malignant cells as 3D spheroids was associated with switch to glycolysis metabolism and up-regulation of survival signals (e.g. Mcl1, Hsp70 et.c.). We conducted a genome-wide CRISPR screen in MCF7 and MDAMB231 2D cultures and identified several known and previously underappreciated BrCa essential genes; we are currently using the insight obtained by these molecular analysis to conduct focused CRISPR screens in our 3D cultures, to validate the genetic vulnerabilities observed in 2D conditions and to determine the mediators of malignant growth and drug resistance in 3D conditions. This approach can reveal novel and previously underappreciated therapeutic targets for drug-resistant BrCa.

Citation Format: Dhimolea E, De Matos Simoes R, Awate P, Tang H, Culhane A, Mitsiades C. High-throughput pharmacogenomic platform to functionally probe breast cancer vulnerabilities in the context of the 3-dimensional tumor microenvironment [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-03-07.