Bone marrow LysM-Cre mediated TFEB overexpression significantly affected the response of bone marrow-derived macrophages to tumor material, decreasing both the expression of typical M2-like macrophage associated genes and inflammatory response genes.

In order to examine the effects of TFEB overexpression in macrophages on anti-tumor immunity, 8 week-old, female LysM-Double-Cre x TFEB transgene+ (TFEBtg) and LysM-Double-Cre x TFEB transgene- (WT) mice were sacrificed and their bone marrow was differentiated into macrophages using recombinant M-CSF. After one week of differentiation, macrophages were replated and treated with either DMEM only or DMEM supplemented with tumor-conditioned medium (serum-free DMEM supernatant exposed to EO771 tumor cells) and tumor lysate for 24 hours. Macrophages were then collected and gene expression was analyzed using whole genome microarray. TFEBtg macrophages expressed significantly less typical M2-like macrophage genes, such as Arg1 and IL-10, than WT macrophages when exposed to tumor-conditioned medium and lysate. TFEB overexpression also decreased the expression of pro-inflammatory genes such as IL-1β, IL-6, and NLRP3 as well as genes involved in PGE2 synthesis, such as COX2, HIF-1α, MIF, and cPLA2. RT-qPCR was used to confirm the expression of the genes listed and many others. Ingenuity Pathway Analysis was also performed in order to analyze the expression of genes clustered by functional relationships. Mice treated to increase TFEB expression demonstrated decreased breast tumor growth. Four-condition, one-color experiment. Microarray analysis of LysM-Double-Cre x TFEB transgene+ and LysM-Double-Cre x TFEB transgene- mouse gene expression was performed on bone marrow-derived macrophage RNA. 3 biological replicates per each experimental group.