MnTE-2-PyP Inhibits Stat6 Activation and Reprograms Pro-Tumor M2 Macrophages to Anti-Tumor M1

Macrophages (Mφ) have high plasticity and can polarize along a spectrum of two extremes, the pro-inflammatory M1 Mφ and the immuno-suppressive M2 Mφ. In highly aggressive triple negative breast cancer, tumor associated Mφ (TAM) have a pro-tumor M2-like phenotype and are associated with disease progression and decreased survival. Thus, it is critical to develop strategies to reprogram TAM away from pro-tumor M2 Mφ toward anti-tumor M1 Mφ. Recently, a lipid peroxide scavenger displayed some efficacy to selectively inhibit M2 Mφ markers and cytokines. Little is known about the redox status of M1 and M2 Mφ, which could shed light on this phenomena. Our hypothesis is pro-tumorigenic M2 Mφ possess a distinct profile of pro- and anti-oxidant enzymes resulting in a different sensitivity to ROS modulation versus the anti-tumor M1 Mφ. Our results show M2 Mφ, derived from primary human monocytes, have a significant 20-30% reduction in intracellular ROS levels versus M1 Mφ by DCFH and DHE staining, as well as dramatically reduced extracellular H 2 O 2 by ~70%. These changes are due in part to a combination of decreased ROS producing enzymes, Nox2, Nox5, and the Nox accessory protein CYBA, and increased ROS scavenging enzymes, catalase, Gpx1/4, and Cu/ZnSOD, in M2 Mφ. These data suggest M2 Mφ have increased ROS metabolism versus M1 Mφ, which may provide an advantage in the oxidative tumor microenvironment. Additionally, increasing ROS with exogenous H 2 O 2 , lead to increased M2 markers and decreased M1 markers implicating the oxidative tumor microenvironment in promoting a M2-like TAM phenotype. Similarly, reducing the extracellular oxidation by EcSOD re-expression in MB231, a triple negative breast cancer cell line, inhibited the MB231-mediated increase in M2 Mφ markers and decrease in M1 Mφ markers. Treatment with Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE), a small molecule SOD mimetic, shown to have anti-tumor effects, increased M1 Mφ markers, IL-12b and CD86, while decreasing M2 Mφ markers, IL-10, CD163, and CD206. MnTE inhibited the ability of M2 Mφ to inhibit T cell activation and promote cancer cell growth. Activation of Stat6, a key transcriptional regulator of M2 Mφ, is reduced with MnTE treatment providing mechanistic rationale for the inhibitory role of MnTE on M2 Mφ. These studies highlight the pivotal role of ROS during M2 Mφ polarization and provide rationale for antioxidant treatment of breast cancer to reprogram TAM.