PO-274 Development of an in vitro tumour model to evaluate drug efficacy and epithelial mesenchymal transition in triple negative breast cancer

Abstract

IntroductionThe advent of diverse methods for 3-dimensional (3D) cell culture has allowed scientists to address some of the limitations of conventional 2D methods and have been applied to studies concerned with cancer therapy response. A major component of the tumour microenvironment overlooked during drug discovery and cancer studies is the interstitial fluid pressure (IFP).The aim of this project is to study breast cancer cell invasion and responsiveness to chemotherapeutic agents in a more physiologically relevant environment taking into account the effect of IFP and fluid flow.Material and methodsMDA-MB231 breast cancer cells were selected due to their triple negative subtype and were seeded in a dense (80 mg/mL) collagen scaffold, to form an ‘artificial cancer mass’ (ACM). The metabolic activity of the cells in 2D, 3D, static and under flow conditions: 100 and 500 µl/min; IFP: 19 mmHg were evaluated using an Alamar Blue assay. Markers of epithelial-mesenchymal transition (EMT) were determined using qRT-PCR and ΔCt values derived with reference to GAPDH. The responsiveness of the cells to doxorubicin was assessed.Results and discussionsMDA-MB231 cells exhibited a decrease in metabolic activity when cultured in 3D compared to 2D. The 3D/flow environment promoted a switch from an epithelial to a mesenchymal phenotype as evidenced by increased expression levels of Snail (2 fold); vimentin (0.6 fold); MMP1 (0.7 fold); HIF-α (0.2 fold). In contrast, incorporation of IFP in the 3D/flow system was associated with decreased expression levels of vimentin (0.25 fold) and HIF-α (0.56 fold) compared to cells grown in static 3D whilst MMP 14 expression increased (0.5 fold) and snail remained unchanged. 3D/flow was also associated with a reduction in cadherin 11 mRNA levels. Reports have suggested that cadherin-11 has a pro-apoptotic role; analysis of apoptotic markers is planned in order to investigate this finding further.Cells grown in 3D culture were less responsive to doxorubicin treatment compared with those grown in 2D monolayer. When MDA-MB231 cells were grown in 3D/flow/pressure and treated with doxorubicin 2.5 µM, the metabolic activity measured using Alamar blue was 41% lower than for MDA-MB231 cells cultured in 3D static conditions.ConclusionThe results obtained in this study show that the metabolic activity and drug responsiveness of the cancer cell line MDA-MB231 is reduced when cells are grown in 3D. The implementation of flow and IFP on 3D cell cultures promotes EMT of MDA-MB231 cells.