The Role of Macrophages in The Chronic Activation of Cancer Cells and Tumour Progression

Abstract

Tumour microenvironment cells play important roles in tumour growth and progression. Tumour-associated macrophages (TAM) are one of the most abundant inflammatory leukocytes in the tumour microenvironment and known drivers of carcinogenesis. Therefore, investigating how in vitro differentiated macrophages affect cancer cells' biological behaviour in different co-culturing conditions would help in understanding cancer biology and offer insight into novel treatment strategies. Human monocytic Thp-1 cells differentiated by PMA were used as an in vitro alternative model for human peripheral-blood macrophages in this project. As several studies used the co-culture model to test the roles of immune cells in cancer cells, we used PMA-differentiated macrophages to be co-cultured with A549 lung cancer cells under different conditions. Those published papers used co-culturing techniques to investigate cancer cells' behaviour upon short-term coculturing with macrophages, which differs from in vivo tumour biology that arises from several weeks of interactions with tumour microenvironment cells. In the first part of this project, we assessed gene expression and functional changes in A549 cells co-cultured with macrophages for a short period of time (3 days). The results showed that our differentiated macrophages in the co-cultured transwell model system induced gene expression, proliferation, and epithelial-mesenchymal transition in A549 cells. The second part of this project was established to test the hypothesis that long term crosstalk of cancer cells with macrophages could cause chronic changes in cancer hallmarks that remained after removing the stimuli (macrophages) due to information stored in cancer cell memory about responding to the stimulus. To investigate this hypothesis, we rested A549 cells in single culture after 3 days or 30 days of co-culturing with macrophages. Our results show that co culturing A549 cells with macrophages for a long time resulted in a sustained mesenchymal transition state and induced proliferation after removing macrophages in long-programmed A549 cells. However, resting A549 cells in single culture after short-term co-culture with macrophages returned them to an epithelial state, while proliferation remained induced in short programmed cells. Further RNA sequencing data analysis revealed the similarities between "long-programmed" and "CO 3 days" cells in their differential gene expression pattern and the activation of cancer progression related pathways. However, no significant differences were found between short programmed and parental A549 cells except for proliferation-related genes expression, which was remained-induced in short programmed A549 cells which reflected in their proliferation functional assays.