ABSTRACT Tumor has been described as the wounds that do not heal. The two share some common features, such as loss of polarized tissue structure and chronic inflammation. We showed that disruption of tissue polarity induced macrophage infiltration. However, little is known of how disruption of epithelial cell polarity at early stage of breast cancer development induces macrophage infiltration. We have identified the RAR-related orphan nuclear receptor α (RORα) as a potent tumor suppressor by analyzing global gene expression profiles in polarized and non-polarized mammary epithelial cells. Our recent findings show that RORα inhibits ROS generation and macrophage infiltration in the syngeneic mouse mammary tumor model. These results suggest that RORα is a potent suppressor of macrophage infiltration in mammary epithelial cells. We found that knockdown of RORα significantly induced ROS production in mammary epithelial cells. Reactive oxygen species (ROS) are the driver of cancer progression and critical regulator of the NF-κB pathway. Based on these novel findings, the central hypothesis of our proposal is that downregulation of RORα in non-polarized breast cancer cells increased ROS generation in mitochondria, thereby inducing NF-κB and macrophage infiltration. We integrate high-throughput metabolic analysis, a novel 3D co-culture system, and global gene expression profiling to delineate mechanisms by which RORα inhibits ROS production and macrophage infiltration. The long-term goal of this proposal is to define the impact of the RORα/ROS axis in mediating mammary epithelial cell-macrophage crosstalk and in regulating breast cancer progression. We have proposed following specific aims to test the hypothesis: Aim 1. To elucidate the molecular mechanisms by which RORα reduces ROS levels and NF-κB activity in polarized mammary epithelial cells; Aim 2. To determine how reduced RORα expression in non-polarized breast cancer cells induces macrophage infiltration and M2 polarization; Aim 3. Define the impact of RORα in suppressing breast cancer formation and metastasis. The proposed study is high impact for its inherent scientific importance and its translational potential. This study will elucidate the molecular mechanism by which disruption of tissue polarity induces macrophage infiltration/differentiation. Determining roles of RORα in reducing ROS generation and inhibiting NF-κB activation may identify a novel strategy to inhibit breast cancer development and progression.