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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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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:Human WhartonM-bM-^@M-^Ys jelly stem cells (hWJSCs) are derived ethically in large amounts from the umbilical cord matrix. Besides their differentiation capabilities, WJSCs also display a notable lack of ability to form teratoma. hWJSCs have been shown to exert immunomodulatory effects and have recently been reported to kill or diminish cancer cell growth. These characteristics are important considerations for their use in cell therapy. In this transcriptome analysis, hWJSCs were profiled using Affymetrix DNA microarrays and compared to a panel of human stem cells and stromal cells. Although hWJSCs are multipotent, they expressed very low levels of the majority of stem cell markers, including POU5F1, NANOG, SOX2 and LIN28. BIRC5 has recently been shown to be required for teratoma formation in SCID mice. The lower levels of BIRC5 expression in hWJSCs compared to hESCs and the very low levels of stem cell markers might account for hWJSCs inability to form teratomas. IL12A which is known to be associated with the induction of apoptosis, was amongst the several cytokines identified to be significantly upregulated in hWJSCs. The ability of hWJSCs to compliment the host immune responses was further highlighted with the GO Biological Process analysis showing high association with immune system, chemotaxis and cell death. The ability to modulate immune responses confers hWJSCs an additional advantage in stem cell therapy and potentially allows hWJSCs as a form of treatment for cancer and immune disorders. In summary, the transcriptome profile of hWJSCs has provided indications on the genetic basis for their biological characteristics in immunomodulatory response, anti-cancer effects, and the lack of teratoma formation. This 'meta-analysis' dataset contains hWJSCs and hFC that were generated by our group, and selected samples available from the public GEO database (human mesenchymal stem cells, umbilical vein endothelial cells, normal stroma, tumour stroma, fibroblast).

Project description:Background: In several types of malignancies, especially EBV-associated nasopharyngeal carcinomas, high Galectin-9 (Gal-9) expression is indicative of an aggressive tumor phenotype. The contribution of Gal-9 to the oncogenesis of B-cell lymphomas (BCLs) has not yet been investigated. Methods: The expression of Gal-9, STING, and EBNA1 was measured by immunohistochemical (IHC) staining on tumor sections from 66 BCL patients. Artificial EBV infection of normal primary B cells in vitro was used as an experimental model. The dynamic changes of the transcriptome of EBV-infected B cells undergoing transformation were investigated by bulk RNA-sequencing and bioinformatics analysis. The oncogenic role of Gal-9 was investigated in vitro by addition of recombinant Gal-9 to EBV-infected primary B-cells, and growth assays. The underlying molecular mechanisms were investigated by immunoblotting and immunofluorescent (IF) staining, CHIP and luciferase reporter assays. Results: In clinical specimens of BCLs, Gal-9 expression was significantly associated with tumor stage, latent EBV infection and abundance of the viral latent protein EBNA1. Looking at the transcriptome changes occurring in vitro during EBV-driven B-cell transformation, we could identify a series of genes undergoing long term activation and remaining highly expressed in mature LCLs. The Gal-9 gene is one of them. Its expression is enhanced during the transformation process at the mRNA level and even more at the protein level. This up-regulation results at least in part from its transactivation by EBNA1 which can bind its promoter. Reciprocally, we find that addition of extra-cellular Gal-9 promotes B-cell transformation and establishment of the latent phase of EBV-infection. Concomitantly, extra-cellular Gal-9 blocks STING signaling and enhances STAT3 phosphorylation. Inhibition of Sting signaling or STAT3 phosphorylation blocks B-cell transformation, even in the presence of Gal-9. Conclusion: our data unveil a role of amplification and acceleration for Gal-9 in the process of EBV-driven B-cell transformation. This process might be relevant to the pathogenesis of EBV-associated BCLs.

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

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