Inducible nitric oxide synthase-derived extracellular nitric oxide flux regulates proinflammatory responses at the single cell level

The role of nitric oxide (NO) in cancer progression has largely been studied in the context of tumor NOS2 expression. However, pro- versus anti-tumor signaling is also affected by tumor cell-macrophage interactions. While these cell-cell interactions are partly regulated by NO, the functional effects of NO flux on proinflammatory (M1) macrophages are unknown. Using a triple negative murine breast cancer model, we explored the potential role of macrophage Nos2 on 4T1 tumor progression. The effects of NO on macrophage phenotype were examined in bone marrow derived macrophages from wild type and Nos2(−/−) mice following in vitro stimulation with cytokine/LPS combinations to produce low, medium, and high NO flux. Remarkably, Nos2 induction was spatially distinct, where Nos2(high) cells expressed low cyclooxygenase-2 (Cox2) and vice versa. Importantly, in vitro M1 polarization with IFNγ+LPS induced high NO flux that was restricted to cells harboring depolarized mitochondria. This flux altered the magnitude and spatial extent of hypoxic gradients. Metabolic and single cell analyses demonstrated that single cell Nos2 induction limited the generation of hypoxic gradients in vitro, and Nos2-dependent and independent features may collaborate to regulate M1 functionality. It was found that Cox2 expression was important for Nos2(high) cells to maintain NO tolerance. Furthermore, Nos2 and Cox2 expression in 4T1 mouse tumors was spatially orthogonal forming distinct cellular neighborhoods. In summary, the location and type of Nos2(high) cells, NO flux, and the inflammatory status of other cells, such as Cox2(high) cells in the tumor niche contribute to Nos2 inflammatory mechanisms that promote disease progression of 4T1 tumors.