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High-throughput screening and proteomic characterization of compounds targeting myeloid-derived suppressor cells

ABSTRACT: Myeloid-derived suppressor cells are a heterogeneous cell population of incompletely differentiated immune cells. They are known for suppressing T cell activity and are implicated in multiple chronical diseases, which makes them an attractive drug target for the pharmaceutical industry. Here, we differentiated mouse MDSC from a progenitor cell line and used quantitative (phospho)proteomics to quantify more than 7,000 proteins and phosphorylation sites that enable the characterization of MDSC on a molecular level. Based on this differentiation protocol, we investigated the effects of the well-studied MDSC drugs Entinostat and Mocetinostat on a proteomewide level and established a high-throughput drug screening platform. We assessed the effects on T cell proliferation and INF-γ secretion of ~21,000 small molecules in a MDSC/T cell coculture setup. The most promising candidates were further validated in a screening setup using human MDSC. Finally, a proteomics experiment showed the significant upregulation of several proteins associated with the reduction of reactive oxygen species, suggesting the potential mode of action of this compound.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Johannes Krumm

LAB HEAD: Bernhard Kuster

PROVIDER: PXD043407 | Pride | 2023-08-15

REPOSITORIES: Pride

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			Action	DRS
	03494_BA1_P040345_S00_I00_U01_R1_TMT10.raw	Raw
	03494_BA2_P040345_S00_I00_U09_R1_TMT10.raw	Raw
	03494_BB1_P040345_S00_I00_U02_R1_TMT10.raw	Raw
	03494_BB2_P040345_S00_I00_U10_R1_TMT10.raw	Raw
	03494_BC1_P040345_S00_I00_U03_R1_TMT10.raw	Raw

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Project description:The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naive neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC). In the current study, a transcriptomics approach was used in mice to compare these cell types. Our data show that the three populations of neutrophils are significantly different in their mRNA profiles with NN and G-MDSC being more closely related to each other than to TAN. Structural genes and genes related to cell-cytotoxicity (i.e. respiratory burst) were significantly down-regulated in TAN. In contrast, many immune-related genes and pathways, including genes related to the antigen presenting complex (e.g. all six MHC-II complex genes), and cytokines (e.g. TNF-a, IL-1-a/b), were up-regulated in G-MDSC, and further up-regulated in TAN. Thirteen of the 25 chemokines tested were markedly up-regulated in TAN compared to NN, including striking up-regulation of chemoattractants for T/B-cells, neutrophils and macrophages. This study characterizes different populations of neutrophils related to cancer, pointing out the major differences between TAN and the other neutrophil populations. Various types of myeloid cells have been shown to promote tumor progression by direct immune suppression and by production of angiogenic factors, matrix-degrading enzymes, or growth factors. In untreated tumors, neutrophils have been reported to produce angiogenic factors and matrix-degrading enzymes, support the acquisition of a metastatic phenotype, and suppress the anti-tumor immune response. Neutrophils, like all other leukocytes, move into tissues from the blood under the influence of specific chemokines (e.g. KC/CXCL-1, MIP-2a/CXCL-2 and GCP-2/CXCL-6), cytokines (e.g. TNFa and IFN-x), and cell adhesion molecules located on their own surface (e.g. CD11b) and on the surface of endothelial cells (e.g. selectins, ICAM-1 and PECAM-1). When they traffic into tumors, they are referred to as TAN. In mice, TAN can be defined by the specific surface markers CD11b and Ly6G with low expression of macrophage markers such as F4/80. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immune suppressive cells that are produced excessively in cancer. They comprise at least two subsets -granulocytic (Ly6G+, G-MDSC) and monocytic cells (Ly6C+, M-MDSC), potentially with different immunosuppressive properties. It has been previously shown that MDSC can enter tumors and differentiate to mature macrophages (TAM) or neutrophils (TAN). However, since no definitive markers have been established, it is unknown whether intratumoral N2 neutrophils (N2 TAN) are granulocytic MDSC from spleen that are attracted to the tumor or if they are typical blood-derived neutrophils that are then converted to an N2 phenotype by the tumor microenvironment, specifically by the high local concentrations of TGF-b. The purpose of this study was to use a transcriptomics approach to gain further information about TANs by comparing the RNA profile of these cells to naive bone-marrow neutrophils (NN) and to the granulocytic fraction of myeloid derived suppressor cells (G-MDSC). We examined which pathways and gene-groups varied amongst these 3 populations of neutrophils and performed a detailed analysis on pathways related to the main functions of neutrophils, such as respiratory burst, granule proteins, phagocytosis, apoptosis, structural genes, antigen presentation and specific immune effects. Our data defines TAN as a unique population of neutrophils, quite distinct from both NN and G-MDSC.

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High-throughput screening and proteomic characterization of compounds targeting myeloid-derived suppressor cells

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