Abstract 4497: Delineating the effects of different levels of nitric oxide produced by "M1" stimulated macrophages on tumor rejection and response to therapy
Authors: Somasundaram Veena , Basudhar Debashree , Palmieri Erika M. , Cheng Robert Y. , Ridnour Lisa A. , McVicar Daniel W. , Wink David A. 
Published: August 2018

Nitric Oxide (NO) was historically known to be immunosuppressive in cancers by inhibiting T-cell function and endothelial activation. This is relevant however only in the aberrant, dysfunctional tumor vasculature. Recently, local low dose irradiation was found to cause the differentiation of NOS2+ macrophages in the tumor microenvironment, that then normalized the vasculature and allowed T-cell infiltration, subsequent tumor rejection and prolonged survival in mouse models of pancreatic cancer (Klug et al., 2013). It was later shown that high doses of NO inhibited normal vascularization while at low doses, NO induced the expression of endothelial cell adhesion molecules and favored T-cell infiltration (Sektioglu et al., 2016). Previously, we found that murine macrophages can be activated by different cytokines/LPS to produce different levels of NO in the extracellular space (Espey et al., 2000). In the current study, we used these cytokine/LPS combinations to stimulate macrophages to produce distinct NO levels and delineate their intracellular effects. The levels of NO in bulk solution in vitro was determined using various methods for measuring NOS2 protein and various products of the NO/O2 reaction using Griess assay and nitrosation of fluorescent dyes like diamino fluorescein (DAF) and diamino naphthalene (DAN). At the single cell level, microscopy and flowcytometry were employed to assess NOS2 protein levels. Status of oxidative phosphorylation (OxPhos) and glycolytic commitment in the stimulated macrophages was studied using the Seahorse bioanalyzer. The high NOS2 induction by IFNγ/LPS followed by IFNγ/TNFα and then IFNγ/IL1β as seen in the bulk environment was spatially and temporally regulated with IFNγ/LPS inducing NOS2 in as early as 4h earlier and in more number of cells while IFNγ/TNFα starts inducing NOS2 at 8h, in fewer cells and IFNγ/IL1β only induces very low NOS2 although in all the cells. These fluxes could be directly correlated to changes in bioenergetics with IFNγ/IL1β activating OxPhos above resting macrophages while IFNγ/LPS causes glycolytic commitment. The NO levels also regulate expression of cell adhesion molecules on endothelial cells. The NOS2 induction is spatially and temporally regulated and NO fluxes were also cell type and species specific although the trend of IFNγ/LPS &gt; IFNγ/TNFα &gt; IFNγ/IL1β is maintained across the macrophage types analyzed. We find that these different levels have distinct effects on macrophage metabolism and endothelial activation. Hence, we now have insights into which NO flux can help improve response to cancer immunotherapy versus cause immunosuppression.

Citation Format: Veena Somasundaram, Debashree Basudhar, Erika M. Palmieri, Robert Y. Cheng, Lisa A. Ridnour, Daniel W. McVicar, David A. Wink. Delineating the effects of different levels of nitric oxide produced by "M1" stimulated macrophages on tumor rejection and response to therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4497.