Investigation of the in vitro use of HL-60 cells induced at different maturation levels as myeloid-derived suppressor cell (MDSC)-like

Objective: HL-60 cell line is an acute myeloid leukemia cell line and is used as a model in studies at different myeloid maturation levels. These cells are known to differentiate into granulocyte and/or monocyte/macrophage-like cells by some factors. Myeloid-derived suppressor cells (MDSC) are myeloid cells that leave the bone marrow at different maturation levels as a result of chronic inflammation and join the peripheral circulation and can perform immune regulation. Although these cells have a key role in many pathologies, there is no cell line that can be used in mechanistic studies. In this study, the effects of all-trans retionic acid (ATRA), 1a,25-dihydroxyvitaminD3 (D3), secreted factors from MDA-MB-231 or MDA-MB-468 cells on MDSC-like transformation on HL-60 cells were investigated. Method: In order to evaluate MDSC-like chracteristics of HL-60 cells, were stimulated with ATRA and D3 for 24, 48 and 96 hours and/or were co-cultured with MDA-MB-231 and MDA-MB-468 conditioned medium (CM) for 72 hours to 8 days. Stimulated cells were evaluated by Giemsa staining for morphological changes; surface markers of CD11b, CD11c, CD14, CD15, CD16, CD33, CD40, CD62L, CD66b, CD70, CD114, TRAIL, TLR4, HLA-DR and Lox-1 were evaluated by flow cytometry; stimulated HL-60 cells on T cell proliferation were evaluated by CFSE assay; ROS (reactive oxygen species) and NO (nitric oxide) production capacities were investigated via flow cytometry. NOX2, MPO, COX2, IDO1, NOS2, ARG1, ARG2 mRNA trancription levels were evaluated by qRT-PCR. Results and Conclusion: ROS production, NOX2 and MPO mRNA expression levels were increased in response to ATRA-stimulation. CD11b expression increased in all conditions compared to unthereated HL-60, CD14 positivity increased especially in D3-stimulated conditions. However, no difference was observed in NO production and NOS2 mRNA level in cells stimulated with ATRA and D3. CD114 and Lox-1 expression were detected on CD11bhigh in MDA-MB-231 or MDA-MB-468 CM threated HL-60 cells. Especially, LOX-1 expressio was increased in response to CM from MDA-MB-231/MDA-MB-468 on day 8. HL-60 cell lines was exhibit granulocytic (G)-MDSC-like phenotype when stimulated with MDA-MB-231/MDA-MB-468 CM. Lox-1 surface marker which is critial for G-MDSC phenotype, upregulated successfully with CM induction.