DIFFERENTIATION OF MACROPHAGES BY EXTRACELLULAR VESICLES DERIVED FROM HYPOXIA-CULTURED BREAST CANCER CELLS

Abstract

A great part of the total mass in a tumor microenvironment comprises recruited macrophages,which may act in pro- or anti-tumoral responses depending on its interaction with the microenvironment. M2 macrophages are essential for metastatic processes, contributing to a pro-angiogenic and invasive context. In tumor cells, particularly under stress situations such as hypoxia, the communication between cells from the microenvironment is mediated by extracellular vesicles (EV), which may trigger the differentiation of tumor-associated macrophages (TAM). Considering that triple-negative breast cancer is a predominant illness in Brazilian women, in-depth investigations of its basic biology may aid in the discovery of new therapeutic targets. In this project, we propose the investigation of macrophage differentiation mediated by hypoxic extracellular vesicles from breast cancer cells. To do so, we intend to use cell lines for monocytes THP-1 and triple-negative breast adenocarcinoma MDA-MB-231. Monocyte to macrophage differentiation will be induced by the addition of conditioned media containing hypoxic EVs from breast cancer cells and analysed in timelapse optical microscopy. Positive control comprises phorbol-12-myristate-13-acetate (PMA)treatment. Monocyte and macrophage phenotyping will be performed by flow cytometry and RT-qPCR. Co-culture assays will be performed to study the interaction between the two cell lines, and zymography of the cell culture assays will be performed to identify matrix metalloprotease (MMP). We hypothesize that monocytes will adhere in co-culture with MDA-MB-231 cells, with increased activation of MMP-9, and that hypoxic EVs will optimize macrophage differentiation to distinct subtypes.