Galectin-1 induces a tumor-associated macrophage phenotype and upregulates indoleamine 2,3-dioxygenase-1

Abstract

Galectins are a group of carbohydrate-binding proteins with a presumed immunomodulatory role and an elusive function on antigen-presenting cells. Here we analyzed the expression of galectin-1 and found upregulation of galectin-1 in the extracellular matrix across multiple tumors. Performing an in-depth and dynamic proteomic and phosphoproteomic analysis of human macrophages stimulated with galectin-1, we show that galectin-1 induces a tumor-associated macrophage phenotype with increased expression of key immune checkpoint protein programmed cell death 1 ligand 1 (PD-L1/CD274) and immunomodulator indoleamine 2,3-dioxygenase-1 (IDO1). Galectin-1 induced IDO1 and its active metabolite kynurenine in a dose-dependent manner through JAK/STAT signaling. In a 3D organotypic tissue model system equipped with genetically engineered tumorigenic epithelial cells, we analyzed the cellular source of galectin-1 in the extracellular matrix and found that galectin-1 is derived from epithelial and stromal cells. Our results highlight the potential of targeting galectin-1 in immunotherapeutic treatment of human cancers.

Keywords: Cancer; Immunology.

Graphical abstract

Figure 1

Gal-1 is highly expressed in the tumor microenvironment and associated with decreased survival (A) RNA sequencing data for LGALS1 (galectin-1), LGALS3 (galectin-3), and LGALS8 (galectin-8) in a number of human tumor samples (red boxes) and the corresponding normal samples (blue boxes). (B) Immunofluorescent staining of gal-1, gal-3, and gal-8 in oral squamous cell carcinomas and normal human oral epithelia. (C) Immunohistochemistry of Gal-1 of pancreatic adenocarcinomas and head and neck squamous cell carcinoma. Specimens from three different patients are shown for each cancer. Images were extracted from the Human Proteome Atlas (https://www.proteinatlas.org). In all images Gal-1 is expressed predominantly in the stroma, although expression in the epithelial compartment is also observed in some cancers; a feature that is especially prominent in one of the cases of kidney adenocarcinomas. (D) Overall survival analysis in head and neck cancer and pancreatic cancer. The survival analysis was done at GEPIA with default settings (http://gepia2.cancer-pku.cn). Data in (A and D) were obtained from the online database GEPIA. CHOL, cholangial carcinoma; DLBC, lymphoid neoplasm diffuse large B-cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; PAAD, pancreatic adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; THYM, thymoma.

Figure 2

Transcriptional effects of Gal-1 on different antigen-presenting cells (A) Six-day protocol for isolation and cytokine stimulation of monocytes for differentiation into M1-like MΦs, M2-like MΦs, and DCs. Stimulation with or without Gal-1 (300 ng/mL, 24 h) was performed on day 5 following RNA extraction and processing for RNA sequencing. (B) Principal-component analysis of transcriptomic data in unstimulated or Gal-1-stimulated M1-like MΦs, M2-like MΦs, and DCs. (C) Functional enrichment analysis of enriched Gene Ontology terms in Gal-1-stimulated M1-like MΦs, M2-like MΦs, and DCs. (D) Heatmap of the most regulated genes in M1-like MΦs, M2-like MΦs, and DCs with or without Gal-1.

Figure 3

Gal-1 modulates the proteomic landscape of M2-like MΦs toward a tumor-associated macrophage phenotype (A) Overview of the analytical strategy for TMT-based proteomic and phosphoproteomic analysis of M2-like MΦs. M2-like MΦs (n = 16, 1 donor, 2 replicates for each time point) were stimulated with or without Gal-1 (300 ng/mL) for 0 min, 5 min, 6 h, or 12 h. Subsequently, cells were lysed and labeled with TMTpro 16-plex reagents and pooled. A small fraction of pooled sample was reserved for proteome analysis, and the rest was enriched by Ti-IMAC for phosphoproteome analysis. Proteome and phosphoproteome samples were then fractionated and analyzed by LC-MS/MS. (B) Soft clustering of proteins in Gal-1-stimulated samples. Identified proteins (n = 8337) were clustered into six different clusters based on the ratio distribution across all time points. (C) Functional enrichment analysis with proteins extracted from clusters 4 and 6. (D) Network plot of the most regulated proteins and assigned GO terms. The size of the central nodes (gray) indicates the number of proteins represented, and the color of protein nodes indicates log2 fold changes. (E) Dynamic expression of regulated proteins involved in JAK/STAT signaling in Gal-1-stimulated M2-like MΦs.

Figure 4

Gal-1 induces expression of IDO1 (A) Dynamic expression of IDO1, PD-L1/CD274, and IL4I1 in Gal-1-stimulated M2-like MΦs. Left: Expression of IDO1 and PD-L1/CD274 in TMT-labeled samples from donor 1. Right: Expression of IDO1, PD-L1/CD274, and IL4I1 in label-free samples from the other three donors. (B) Expression of IDO1 in M2-like MΦs stimulated with or without Gal-1 (300 ng/mL or 600 ng/mL) and/or lactose (lac) (100 mM) for 6 h, 12 h, 16 h, or 24 h. (C) Kynurenine formation in M2-like MΦs stimulated with or without Gal-1 (300 ng/mL) and/or lac (100 mM). Data are represented as mean +SEM. (D) Kynurenine formation over 24 h in M2-like MΦs stimulated with or without Gal-1 (300 ng/mL) and/or lac (100 mM).

Figure 5

Gal-1 alters the signaling events in M2-like MΦs and activates JAK/STAT signaling (A) Phosphoproteomic analysis of M2-like MΦs stimulated with or without Gal-1 (300 ng/mL) for 0 min, 5 min, 6 h, or 12 h identified 17,283 phosphosites that group into six clusters based on the ratio of distribution over all time points. (B) Functional enrichment analysis of GO terms assigned to phosphosites in cluster 4. (C) Map of all phosphosites and assigned proteins in the JAK/STAT signaling pathway with a significant change at any time point. Colors of nodes indicate the log2 fold change in proteins and phosphosites for the different time points. (D) IDO1 protein expression in M2-like MΦs stimulated with IFNγ (20 ng/mL), Gal-1 (300 ng/mL), JAK/STAT inhibitor tofacitinib (1 μM), ruxolitinib (1 μM), or lactose (100 mM) as indicated for 6 h. (E) Kynurenine formation in M2-like MΦs stimulated with IFNγ (20 ng/mL), ruxolitinib (1 μM), tofacitinib (1 μM), or Gal-1 (300 ng/mL) as indicated for 6 h.

Figure 6

Gal-1 is expressed in the tumor microenvironment in response to cross talk between transformed epithelial cells (A) Immunofluorescent staining of Gal-1/K10 in WT, hRAS-overexpressing (OE), and P53 KO organotypic models. Gal-1 (green), keratin-10 (K10) (red), and DAPI (blue). (B) Enlargement of images indicated by white boxes and fold change in the Gal-1⁺ tissue area. Data are presented as mean intensities of the triplicates. (C) Graphic depiction of the proposed model for the Gal-1-JAK/STAT-IDO1 axis inducing the transition of M2-like MΦs and M1-like MΦ toward TAM-like phenotype. Increased levels of Gal-1 lead to activation of JAK/STAT signaling through yet-unidentified receptor binding/activation. In turn, this induces expression of IDO1, PD-L1/CD274, and a number of cytokines (IL-1α, IL-1β, IL-8, IL-10) acting in a feedback loop to enhance pro-tumorigenic induction of tolerance in M2-like MΦs.