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Galectin-1 upregulates IDO1 and PD-L1 and induces a tolerogenic tumor-associated M2 macrophage phenotype

ABSTRACT: Galectins are a group of carbohydrate binding proteins with immunomodulatory functions. While the effects of galectins on the T cell compartment are well described, the interactions between galectins and antigen-presenting cells (APCs) remain elusive. Here we find increased expression of galectin-1(gal-1) in the stroma of a large selection of cancers, and investigating the effect of gal-1 on stroma localized antigen presenting cells, including monocyte derived dedritic cells and M1 and M2 macrophages (M1s and M2s, respectively). Using an in depth and dynamic proteomic and phosphoproteomic analysis of the effect of gal-1 on M2s we show that gal-1 induces global changes in the proteomic and signaling landscape of M2s, consistent with induction of a tolerogenic macrophage phenotype. Specifically, gal-1 induces expression of key immunomodulatory and tumor-associated proteins, including the key immune checkpoint protein, programmed cell death 1 ligand 1 (PD-L1/CD274) and the immunomodulator, indoleamine 2,3-dioxygenase-1 (IDO1). Gal-1 induced IDO1 and its active metabolite kynurenine in a dose dependent manner, an effect that was prevented by JAK/STAT inhibition. Analyzing gal-1 expression in human tumors we find that gal-1 is upregulated across multiple tumors, and in a 3D organotypic model system equipped with genetically engineered tumorigenic epithelial cells, we find that the tumor associated gal-1 is derived from both from epithelial and stromal cells. Our results highlight the potential of targeting gal-1 in immunotherapeutic treatment of human cancers.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Macrophage

SUBMITTER: Zilu Ye

LAB HEAD: Jesper Velgaard Olsen

PROVIDER: PXD034623 | Pride | 2023-08-14

REPOSITORIES: Pride

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			Action	DRS
	20200330_EXPL2_Evo3_ZY_IDO2_10_5mg2_44min_DIA.raw	Raw
	20200330_EXPL2_Evo3_ZY_IDO2_11_5mg3_44min_DIA.raw	Raw
	20200330_EXPL2_Evo3_ZY_IDO2_12_5mp1_44min_DIA.raw	Raw
	20200330_EXPL2_Evo3_ZY_IDO2_13_5mp2_44min_DIA.raw	Raw
	20200330_EXPL2_Evo3_ZY_IDO2_14_5mp3_44min_DIA.raw	Raw

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Project description:Identification of interactors is a major attempt in cell biology. Not only protein-protein but also protein-carbohydrate interactions are of high relevance for signal transduction in biological systems. Here we aim to identify novel interacting binding partners for the β-galactoside-binding proteins Galectin-1 (Gal-1) and Galectin-3 (Gal-3) in context of the eye disease proliferative vitreoretinopathy (PVR). PVR is one of the most common failures after retinal detachment surgeries and is characterized by the migration, adhesion and epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells (RPE) and the subsequent formation of sub- and epiretinal fibrocellular membranes. Gal-1 and Gal-3 bind in a dose- and carbohydrate-dependent manner to mesenchymal RPE cells and inhibit cellular processes like attachment and spreading. Yet knowledge about glycan-dependent interactors of Gal-1 and Gal-3 on RPE cells is very limited, although this is a prerequisite to unravel the influence of galectins on distinct cellular processes in RPE cells. In this approach, we identified by galectin pull-down experiments and quantitative proteomic screening 131 Galectin-3 and 15 Galectin-1 interactors. They mainly play a role in multiple binding processes and are mostly membrane proteins. Here we focused on two novel identified interactors of Gal-1 and Gal-3 in the context of PVR: the low-density lipoprotein receptor LRP1 and the platelet-derived growth factor receptor beta PDGFRB. We observed crosslinking and lattice formation of exogenous Gal-1 and Gal-3 with LRP1/PDGFRB and ITGB1 on the cell surface of human RPE cells. Weaker binding of Gal-1 and Gal-3 on these interactors and no lattice formation on the cell surface was seen, when complex-type-N-glycosylation was inhibited by treatment of the cells with Kifunensine. In conclusion, the identified specific glycoprotein ligands for Gal-1 and Gal-3 give us new insights in the highly specific binding of galectins to dedifferentiated RPE cells and the resulting prevention of PVR-associated cellular events.

Project description:Tumor infiltrating lymphocytes (TILs) play a critical role in modulating the immunoediting features in certain malignancies like triple negative breast cancer (TNBC). Nevertheless, much is still unknown concerning the specific responses of tumors when challenged by lymphocyte infiltration. Based on this void, we conducted a immuno-phenotype comparison using mRNA sequencing between the TNBC cell line MDA-MB-231 and the luminal breast cancer cell line MCF7 after both were co-cultured with activated human T-cells. We found that, though the cytokine-induced immune signature of the two cell lines was similar, MDA-MD-231 cells were able to transcribe IDO1 at a significantly higher level than MCF7 cells. Though no differences occurred in upstream JAK/STAT1 signaling or IDO1 mRNA stability between the two cell lines, stimulation with IFNÎ³ was able to differentially induce IDO protein expression and enzymatic activity in ER- cell lines compared to ER+ cell lines. Further experiments show that 5-aza-deoxycytidine treatment was able to reverse suppression of IDO1 expression in MCF7 cells, suggesting DNA methylation as a potential determinant in IDO1 induction. By analyzing TCGA breast cancer datasets, we discovered subtype-specific mRNA and promoter methylation differences in IDO1, with TNBC/basal subtypes exhibiting lower methylation/higher expression and ER+/luminal subtypes exhibiting higher methylation/lower expression. We confirmed this trend of IDO1 methylation by bisulfite pyrosequencing breast cancer cell lines and an independent cohort of primary breast tumors. Taken together, these findings suggest that IDO1 methylation regulates anti-immune responses in breast cancer subtypes and could be used as a predictive biomarker for IDO inhibitor-based immunotherapy. To determine the immunomodulatory effects of cytokines secreted by activated human T-cells on breast cancer cells, we performed RNAseq analysis in MCF7 and MDA-MB-231 cells, after co-culturing them with normal PBMCs activated with anti-CD3/CD28 antibodies in a contact-independent manner. MDA-MB-231 or MCF7 cells were co-cultured with PBMCs alone or the conditioned-media or a combination of both for 24 hrs, and then total RNA was harvest for RNA-seq analysis.

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Galectin-1 upregulates IDO1 and PD-L1 and induces a tolerogenic tumor-associated M2 macrophage phenotype

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