Tumor cell-derived asymmetric dimethylarginine regulates macrophage functions and polarization

Asymmetric dimethylarginine (ADMA), which is significantly elevated in the plasma of cancer patients, is formed via intracellular recycling of methylated proteins and serves as a precursor for resynthesis of arginine. However, the cause of ADMA elevation in cancers and its impact on the regulation of tumor immunity is not known.

Three mouse breast cell lines (normal breast epithelial HC11, breast cancer EMT6 and triple negative breast cancer 4T1) and their equivalent 3D stem cell culture were used to analyze the secretion of ADMA using ELISA and their responses to ADMA. Bone marrow-derived macrophages and/or RAW264.7 cells were used to determine the impact of increased extracellular ADMA on macrophage-tumor interactions. Gene/protein expression was analyzed through RNAseq, qPCR and flow cytometry. Protein functional analyses were conducted via fluorescent imaging (arginine uptake, tumor phagocytosis) and enzymatic assay (arginase activity). Cell viability was measured via MTS assay and/or direct cell counting using Countess III FL system.

For macrophages, ADMA impaired proliferation and phagocytosis of tumor cells, and even caused death in cultures incubated without arginine. ADMA also led to an unusual macrophage phenotype, with increased expression of arginase, cd163 and cd206 but decreased expression of il10 and dectin-1. In contrast to the severely negative impacts on macrophages, ADMA had relatively minor effects on proliferation and survival of mouse normal epithelial HC11 cells, mouse breast cancer EMT6 and 4T1 cells, but there was increased expression of the mesenchymal markers, vimentin and snail2, and decreased expression of the epithelial marker, mucin-1 in EMT6 cells. When tumor cells were co-cultured ex vivo with tumor antigen in vivo-primed splenocytes, the tumor cells secreted more ADMA and there were alterations in the tumor cell arginine metabolic landscape, including increased expression of genes involved in arginine uptake, metabolism and methylation, and decreased expression of a gene that is responsible for arginine demethylation. Additionally, interferon-gamma, a cytokine involved in immune challenge, increased secretion of ADMA in tumor cells, a process attenuated by an autophagy inhibitor.

Our results suggest initial immune attack promotes autophagy in tumor cells, which then secrete ADMA to manipulate macrophage polarization favoring tumor tolerance.