Abstract A34: Microenvironment controls breast cancer cells sensitivity to doxorubicin by autophagy inhibition

Abstract

Abstract Drug chemotherapy resistance is the leading cause of death for cancer patients. Most of the studies involving chemotherapy resistance are based on experiments making use of cancer cell lines maintained in two-dimensional (2D) cultures. In these conditions, the influence of extracellular matrix (ECM) and the three-dimensional (3D) microenvironment are not considered, despite clear evidence of their role in genesis and progression of cancer. In the present work, we evaluated the microenvironment control in the MCF-7 cellular response to doxorubicin using the three-dimensional laminin-rich ECM (3D lrECM) cell culture model. Based on cell plating on top of growth factor-reduced Basement Membrane (BD MatrigelTM), the 3D lrECM culture consists of a more physiologically applicable approach for ex vivo analysis of cell behavior. When maintained in a 3D context, these human breast cancer cells showed morphological differences, as viewed by phase contrast microscopy, forming round colonies. The MCF-7 growth rate showed also to be dependent of the cellular culture model used. Despite cancerous, these cells undergo a significant growth arrest on top of laminin-rich ECM. Using the cell viability XTT assays for doxorubicin sensitivity analysis, we observed a drug resistance event in 2D cells treated with low chemotherapy doses (250 and 625 nM of doxorubicin). Thus, while the cell growth on lrECM led to decrease on doxorubicin survival at 60%, the survival rates were almost unchanged (higher than 95% of survival) in monolayer cultures at same treatment conditions. Autophagy, a lysosome-dependent catabolic degradation process, has been implicated in anticancer drug resistance. Moreover, recent reports demonstrated that this cellular phenomenon plays a crucial role as a pro-survival factor in MCF-7 cells treated with epirubicin, a doxorubicin analog. Thus, we investigated whether autophagy is involved in doxorubicin response changes promoted by maintenance in 3D culture. By co-treatment with chloroquine, a pharmacological inhibitor of autophagy, we observed a synergistic effect with doxorubicin only in 2D, with 30% reduction of survival. We also evaluated the chloroquine effect in breast cancer cell response to doxorubicin using specific fluorescent dyes for living, apoptotic and necrotic cells. Inhibition of autophagy increased the cell death percentage in monolayer, by not in 3D cell culture, confirming that ECM abolishes autophagy pro-survival action. Subsequently, the autophagy levels were measured by LC3-II accumulation in both cell culture conditions. Our results suggested that autophagy is significantly inhibited by cellular growth in 3D lrECM. While a clear synergic LC3-II accumulation was observed in 2D cultured cells both treated with doxorubicin and chloroquine, very low levels of LC3-II expression were detected in 3D treated cells. Altogether, our results suggest that autophagy is essential for the tissue microenvironment control of breast cancer cells response to anticancer drugs. Therefore, presence of ECM compounds might prevent the autophagy protective effect from doxorubicin-induced cell death. Financial Support: FAPESP and CNPq Citation Format: Luciana R. Gomes, Alexandre T. Vessoni, Carlos FM Menck. Microenvironment controls breast cancer cells sensitivity to doxorubicin by autophagy inhibition. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A34.