Abstract 1186: Regulation of inducible nitric oxide synthase at the single cell level modulates the inflammatory microenvironment
Authors: Somasundaram Veena , Gilmore Caroline , Palmieri Erika M. , Basudhar Debashree , Heinz Will , Cheng Robert Y. , Ridnour Lisa A. , Lockett Stephen J. , McVicar Daniel W. , Wink David A. 
Published: August 2019

Abstract
The anti-cancer versus pro-tumor behavior of cancer tissues is dependent largely on tumor cell-macrophage interactions in the tumor/inflammatory microenvironment and is regulated by Nitric Oxide (NO). High inducible nitric oxide synthase (Nos2) is associated with poor prognosis in breast cancer. Previously, we found that murine macrophages can be activated by different inflammatory cytokines/LPS to produce distinct NO fluxes (entirely Nos2-derived), suggesting flux-specific biological ramifications of NO. However, the effects of these NO fluxes on M1 phenotype have not been delineated. LC/MS analysis of M1 stimulated, wild type (wt) and Nos2-/- macrophages showed that Nos2 was the only source of citrulline. Metabolic analyses and microscopy showed that flattened cell phenotype characteristic of M1 macrophages and mitochondrial respiration are Nos2 dependent and regulated in a NO flux-dependent manner, while proinflammatory cytokine profile and aerobic glycolysis are Nos2 independent. We show for the first time that induction of Nos2 expression occurs only in specific stimulated cells that also harbor depolarized mitochondria. NO production has been linked to decreased oxygen consumption in hypoxic environments. We utilized a novel, in vitro chamber system that forms cell-generated hypoxic and metabolic gradients in two-dimensions by restricting the diffusive exchange of oxygen and metabolites to a monolayer of cells in a small volume- analogous to diffusion between a capillary and nearby tissue. We investigated interactions between Nos2 in M1-stimulated macrophages and hypoxia and demonstrated that treatment with IFNγ+LPS increases Nos2 expression and alters the magnitude and spatial extent of hypoxic gradients. A modified scratch assay revealed that low doses (1-50μM) of NO increased and high doses (1000μM) inhibited the migratory capacity of 4T1 tumor cells. However, in vivo, Nos2-/- mice did not show difference in primary tumor or metastatic burden compared to wt mice but bone marrow derived macrophages (BMDMs) from wt tumor bearing mice produced significantly lower levels of NO compared to BMDMs from tumor bearing- Nos2-/- mice. In summary, we find that the right flux NO is required to tune the inflammatory microenvironment. Nos2 and citrulline are robust intracellular readouts of extracellular NO flux. Nos2 dependent and independent events cooperate to regulate inflammatory macrophages. Autocrine, single cell effects on metabolism build up to cause paracrine effects including alleviation of hypoxia. 4T1 primary tumor and lung metastasis were not Nos2-regulated but host Nos2 hampered the ability of BMDMs to respond to proinflammatory stimuli hinting at possible systemic effects of Nos2. Hence, to make a reliable prognostic prediction, it is important to know the exact NO flux, which cells within the tumor express Nos2 and what other cells associate with Nos2hi cells.
Citation Format: Veena Somasundaram, Caroline Gilmore, Erika M. Palmieri, Debashree Basudhar, Will Heinz, Robert Y. Cheng, Lisa A. Ridnour, Stephen J. Lockett, Daniel W. McVicar, David A. Wink. Regulation of inducible nitric oxide synthase at the single cell level modulates the inflammatory microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1186.