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

Abstract

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 Nos2high 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 Nos2high 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 Nos2high cells, NO flux, and the inflammatory status of other cells, such as Cox2high cells in the tumor niche contribute to Nos2 inflammatory mechanisms that promote disease progression of 4T1 tumors.

Nitric oxide orchestrates metabolic rewiring in M1 macrophages by targeting aconitase 2 and pyruvate dehydrogenase

“…Indeed, we can rescue MRC with blockade of iNOS during stimulation ( Supplementary Fig. 6E, F) and exposure to low dose of NO affects MRC but not basal OCR 53 . Therefore, we propose that, during stimulation, low NADH/NAD + due to NO-mediated blunting of mentioned enzymatic activities, promotes the accumulation of CI in D form facilitating degradation and favoring full glycolytic commitment.…”

In Vitro Macrophage Immunomodulation by Poly(ε-caprolactone) Based-Coated AZ31 Mg Alloy

“…It is important to note that the coatings containing ZnO nanoparticles, namely PCL-ZnO and PCL-CM-ZnO, predominantly showed a small-rounded morphology typical to unstimulated macrophages as noticed in the absence of LPS (TCPS (−)). However, even though the cellular morphology is seen as a characteristic of the inflammatory response towards the biomaterial [ 81 ], recent data revealed that a modified cell morphology does not necessarily indicate a strongly activated macrophage phenotype [ 110 ].…”

Anin vitrotumorigenesis model based on live cell-generated oxygen and nutrient gradients

“…by the release of NO and PGE2 from the cells 11 . Nos2-expressing cell clumps likely lead to significantly higher local concentrations of NO than could arise when Nos2-expressing cells are scattered and isolated 15 and thus augment this paracrine mechanism.…”

“…11). Macrophages treated with the pro-inflammatory cytokines IFNγ and LPS express higher levels of Nos2 and thus produce more NO, which converts them to a glycolytic pathway and decreases their mitochondrial metabolism and O2 consumption 15 . This effect was observed in REECs as the hypoxic front of treated macrophages was further from the hole than that of untreated cells (Supplemental Fig.…”