Abstract A12: Establishment of myeloid lineage cell line that resembles myeloid-derived suppressive cells

Abstract

Abstract Myeloid-derived suppressive cells (MDSCs) are inflammatory cells that play critical roles in promoting cancer growth and metastasis. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an “MDSC-like” cell line for pharmacological and immunotherapeutic applications. Lysosomal acid lipase (LAL) is a critical lipid enzyme in the metabolic signaling pathway that hydrolyzes cholesteryl esters (CE) and triglycerides (TG) in lysosomes. In mice, lack of LAL in genetically ablated knockout mice (lal-/-) shows systemic expansion of MDSCs, which directly stimulate cancer cell proliferation, and suppress T cell proliferation and impair T cell function. The Affymetrix Genechip microarray assay reveals over-activation of the mTOR signaling pathway in lal-/- MDSCs. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lal-/- mice, we have established wild type (HD1A) and lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Compared with HD1A cells, HD1B cells showed increased glucose concentration, suggesting the enhanced glycolytic metabolic pathway, in which glucose converts into pyruvate. Indeed, the pyruvate concentration was increased in HD1B cells compared with that in HD1A cells. Glycolysis occurs in the cytosol of the cell. Pyruvic acid supplies energy to living cells through the citric acid cycle (TCA) in the mitochondria, which generates NADH for the oxidative phosphorylation (OXPHOS, electron transport pathway) to produce ATP. Aconitase is the rate-limiting enzyme in the TCA cycle. Its activity was doubled in HD1B cells compared with HD1A cells. GLUT (SLC2) family members are the major membrane transporters. Among them, GLUT 1-5 have been well characterized as glucose and/or fructose transporters in various tissues and cell types. Thirteen GLUT proteins have been reported to be expressed in mice (14 in humans). Using the Real-time PCR method, expression of all GLUT members was assessed in HD1A and HD1B cells, in which GLUT3, GLUT6, GLUT8, GLUT12, and CLUT13 were upregulated, while GLUT 5 and GLUT 9 were downregulated in HD1B cells. This supports a concept that the LAL metabolic pathway controls the balance of glucose transportation to fuel the energy need in HD1B cells. In addition, dysfunction of mitochondria in HD1B cells skews toward fission structure, damaged membrane potential, and increased ROS production. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4+ T cell proliferation and function in vitro, and enhanced cancer cell proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an “MDSC-like” cell line. HD1B cells can be used as an alternative in vitro system to study how the lipid metabolic signaling pathway regulates myeloid cell functions. Both HD1A and HD1B cell lines will be useful for identifying pharmacological drugs to suppress MDSCs expansion in multiple inflammation-induced diseases that involve MDSCs. Grant support: This work was supported by National Institutes of Health Grants HL087001 (to H. D.), and CA138759, CA152099 (to C. Y.). Citation Format: Cong Yan, Xinchun Ding, Lingyan Wu, Hong Du. Establishment of myeloid lineage cell line that resembles myeloid-derived suppressive cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A12.