Tumor cell-derived asymmetric dimethylarginine regulates macrophage functions and polarization

Abstract

Background: Asymmetric dimethylarginine (ADMA), which is signifcantly elevated in the plasma of cancer patients, is formed via intracellular recycling of methylated proteins and serves as a precursor for resynthesis of arginine. How‑ ever, the cause of ADMA elevation in cancers and its impact on the regulation of tumor immunity is not known. Methods: 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 fow cytometry. Protein functional analyses were conducted via fuorescent imaging (argi‑ nine 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. Results: 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 expres‑ sion 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 efects 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. Conclusion: Our results suggest initial immune attack promotes autophagy in tumor cells, which then secrete ADMA to manipulate macrophage polarization favoring tumor tolerance.

Description

© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Keywords

Arginine metabolism, Asymmetric dimethylarginine, cancer stem cells, Autophagy, Macrophage polarization

Citation

Chen, YL., Lowery, A.T., Lin, S. et al. Tumor cell-derived asymmetric dimethylarginine regulates macrophage functions and polarization. Cancer Cell Int 22, 351 (2022). https://doi.org/10.1186/s12935-022-02769-7

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