ENGINEERED IN VITRO BREAST CANCER MODELS SHOW PHENOTYPIC DIFFERENTIATION CHARACTERISTICS OF EARLY VS. LATE-STAGE DISEASE

Cancer drug discovery and development is challenged by poor prediction of drug responses in in vitro disease models. Results from clinical trials suggest that just 5% of drugs tested are successful in patients. Currently used disease models such as two-dimensional (2D) cell monolayers and in vivo animal models fail to recapitulate the human tumor microenvironment. Further, disease progression from the non-invasive to metastatic stage needs models that can recapitulate each stage. Hence, there is an unmet need to develop three-dimensional (3D) models that capture natural tumor progression for better understanding of disease biology as well as screening of drug regimens acting on different disease stages. \ud In this work, we have characterized 3D breast microtumors ranging from 150 to 600 μm diameters using non-invasive T47D cells with precise control over physicochemical microenvironmental factors. In this study, we test the hypothesis that the size-controlled microtumors will exhibit differential biochemical features and drug response arising from unique molecular signatures created by variable tumor microenvironments. To test this hypothesis, we studied the physicochemical features such as hypoxia, reactive oxygen species, metabolic activity, and cell cycle status. Additionally, the expression of key regulators of growth/proliferation pathways in breast cancer progression such as estrogen receptor alpha (ER-α) and growth factor receptor/s was studied and efficacy of clinically used inhibitors such as 4-hydroxytamoxifen (4-OHT) was evaluated. \ud The results indicated that large T47D microtumors (600 μm) exhibited traits of clinically advanced tumors such as collective cell migration, mesenchymal marker upregulation, loss of ER-α and endocrine resistance in contrast to the small microtumors (150 μm). Thus, the engineered in vitro models could successfully recapitulate phenotypic differentiation characteristics of early vs. late disease stage in the same non-invasive T47D cells just by precisely controlling the microtumor size, which further regulated the tumor microenvironmental factors. The large microtumors (600 μm) were found to resemble features of advanced stage breast cancer whereas the small microtumors (150 μm) recapitulated features of early stage breast cancer. Hence, such disease stage-specific microtumor models could help in the evaluation of crucial mechanisms in breast tumor progression correlated to tumor size and in the screening of therapeutic candidate/s