Interrogating glioma-M2 macrophage interactions identifies Gal-9/Tim-3 as a viable target against PTEN-null glioblastoma

Abstract

Genomic alteration can reshape tumor microenvironment to drive tumor malignancy. However, how PTEN deficiency influences microenvironment-mediated cell-cell interactions in glioblastoma (GBM) remains unclear. Here, we show that PTEN deficiency induces a symbiotic glioma-M2 macrophage interaction to support glioma progression. Mechanistically, PTEN-deficient GBM cells secrete high levels of galectin-9 (Gal-9) via the AKT-GSK3β-IRF1 pathway. The secreted Gal-9 drives macrophage M2 polarization by activating its receptor Tim-3 and downstream pathways in macrophages. These macrophages, in turn, secrete VEGFA to stimulate angiogenesis and support glioma growth. Furthermore, enhanced Gal-9/Tim-3 expression predicts poor outcome in glioma patients. In GBM models, blockade of Gal-9/Tim-3 signaling inhibits macrophage M2 polarization and suppresses tumor growth. Moreover, α-lactose attenuates glioma angiogenesis by down-regulating macrophage-derived VEGFA, providing a novel antivascularization strategy. Therefore, our study suggests that blockade of Gal-9/Tim-3 signaling is effective to impair glioma progression by inhibiting macrophage M2 polarization, specifically for PTEN-null GBM.

Fig. 1.. PTEN deficiency facilitates macrophage infiltration and M2 polarization.

(A) Immunoblots for PTEN in 23 glioma cell lines. (B) Quantification of relative migration of THP-1 macrophages following stimulation with CM from eight PTEN-null cell lines (U118, U87, U138, U251, SYU354, SF295, U373, and MGR3) and eight PTEN–wild-type (WT) GBM cell lines (SKMG-1, LN229, SYU687, MGR2, LNZ308, GL261, SHG44, and T98). n = 3 biological replicates. (C) Immunoblots showing PTEN expression status in PTEN-deleted (Del) U87/U118 cells after PTEN overexpression (OE) and PTEN-WT SKMG-1/LN229/GL261 cells after PTEN knockout (KO). (D and E) Flow cytometry analysis of CD163 expression in BMDMs treated with CM from PTEN-Del/OE U87 cells and PTEN-WT/KO GL261 cells. (F and G) Immunofluorescence staining of CD86 and CD163 was determined and quantified in intracranial PTEN-WT/KO GL261 and PTEN-Del/OE U87 xenograft tumors. PTEN-WT/KO GL261 xenografts were established in C57BL/6J mice, and PTEN-Del/OE U87 xenografts were established in BALB/c-nu/nu mice. Scale bar, 200 μm. **P < 0.01 and ***P < 0.001.

Fig. 2.. Gal-9, a modulator of macrophage M2 polarization, is abundantly secreted by PTEN-deficient glioma cells.

(A) Transcriptome analyses of up-regulated genes encoding secreted proteins in PTEN-KO LN229 cells. Values are expressed as the fold change between PTEN-KO and PTEN-WT LN229 cells. The top 11 genes (with fold changes of >2.4) were chosen for further validation. (B) qRT-PCR validation of the 11 genes and the TNF gene in PTEN-KO/WT LN229 cells. Values are expressed as the fold change between PTEN-KO and PTEN-WT LN229 cells after normalization to the housekeeping gene GAPDH. (C) Macrophage-inducible factors in PTEN-KO/WT SKMG-1 cells and PTEN-Del/OE U87 cells were detected using antibody microarrays. (D) ELISA analysis of GCM showed Gal-9 secretion up-regulated by PTEN-KO in LN229/SKMG-1 cells and down-regulated by PTEN-OE in U87/U118 cells. **P < 0.01 and ***P < 0.001, Student’s t test. (E and F) Immunoblots of Gal-9 and PTEN or p-AKT expression in the indicated glioma cell lines. (G) Immunoblots for PTEN and Gal-9 in cell lysates of eight short-term patient-derived glioma stem cell lines (GSCs): GSC-1, GSC-#363, GSC-#624, GSC-11, GSC-#354, GSC-#687, GSC-#481, and GSC-#530. (H) Flow cytometry analysis of CD163 and CD206 on THP-1 macrophages treated with U87 CM or recombinant Gal-9 protein (50 ng/ml). (I) Immunoblots showing successful knockdown of Gal-9 in U87 cells by shGal-9. (J) Flow cytometry analysis of CD163 and CD206 on THP-1 macrophages treated with U87 CM or U87 shGal-9#1 CM. (K) Flow cytometry analysis of CD163 and CD206 on THP-1 macrophages treated with U87 shGal-9#1 CM or U87 shGal-9#1 CM supplemented with α-lactose (40 μM).

Fig. 3.. PTEN deficiency–induced Gal-9 production is regulated via the AKT-GSK3β-IRF1 pathway.

(A and B) Immunoblots of Gal-9, AKT, p-AKT^Ser473, and p-GSK3β^Ser9 in PTEN-KO LN229/SKMG-1 cells treated with the AKTi MK2206 at different concentrations and time points. (C and D) Immunoblots of p-GSK3β^Ser9, GSK3β, p-GSK3α^Tyr279, p-GSK3β^Tyr216, IRF1, and Gal-9 in PTEN-KO LN229 cells treated with tideglusib at different concentrations and for different time lengths. (E) Immunoblots of IRF1 and lamin B1 in the cytoplasmic and nuclear components of PTEN-KO LN229 cells treated with different concentrations of tideglusib. (F) Immunoblots of Gal-9 and IRF1 in PTEN-KO LN229/SKMG-1 cells transfected with siIRF1 or control siRNA (siNC). (G) A dual-luciferase reporter system containing the 2.0/1.5/0.5-kb human LGALS-9 promoter DNA fragments was used to predict their transcription activity in indicated glioma cells. The values are expressed as the ratio of Firefly and Renilla luciferase signals. ***P < 0.001, one-way ANOVA. (H) The transcriptional activity of the 0.5-kb human LGALS-9 promoter DNA fragment was detected in PTEN-KO LN229/SKMG-1 cells transfected with siIRF1 or siNC. ***P < 0.001, one-way ANOVA. Band density of immunoblots was determined by ImageJ software, and values represent the expression levels after normalization to the density of GADPH/β-tubulin.

Fig. 4.. scRNA-seq data analysis reveals Gal-9/Tim-3–mediated interactions between glioma cells and macrophages.

(A) Distribution of single cells’ gene expression colored by cell types or samples. On the basis of the expression of marker genes (fig. S8), seven cell types were identified. (B) Cell-cell communication networks for PTEN-null and PTEN-WT samples. The color of the dot represents the cell type, the edge represents the communication between the two cell types whose thickness represents the number of downstream target genes of upstream activated ligand-receptor (LR) pairs, and color represents the direction of cell communication (e.g., the purple edge represents the signals delivered by malignant glioma cells). (C) Stacked violin plots of gene expressions of Gal-9 and Tim-3 in two groups of samples (PTEN-null and PTEN-WT). (D) Gal-9/Tim-3 interaction between glioma cells and macrophages inferred by the multilayer network revealed that macrophage M2 marker genes were regulated by Gal-9/Tim-3 signaling through the downstream pathways and transcription factors (TFs) (e.g., ESR1, ELK1, and STAT1). (E) GO enrichment analysis for the Gal-9/Tim-3 downstream target genes. (F) GSEA enrichment analysis for the ranked gene lists in macrophages according to fold changes of gene expression in the PTEN-null sample compared with PTEN-WT samples.

Fig. 5.. High activation of Gal-9/Tim-3 signaling predicts poor survival of human glioma patients.

Kaplan-Meier survival curves of glioma patients stratified by Gal-9 expression, Tim-3 expression, or the product of Gal-9 and Tim-3 expression (Gal-9 × Tim-3) in the CGGA dataset (A), TCGA dataset (B), and GSE16011 dataset (C). (D) Immunofluorescence staining and quantification of Gal-9 and CD163 expression in human GBM tissues with WT (n = 9) or deleted (n = 9) PTEN gene. Scale bar, 100 μm. **P < 0.01 and ***P < 0.001, Student’s t test. (E) Representative immunofluorescence staining for Gal-9 and Tim-3 in human glioma tissue microarrays. Scale bar, 200 μm. (F) Kaplan-Meier survival curves indicate that high Gal-9 and/or Tim-3 expression correlates with a poor prognosis for glioma patients. Differences in Kaplan-Meier curves were assessed using the log-rank test.

Fig. 6.. Blockade of Gal-9/Tim-3 signaling inhibits macrophage M2 polarization and GBM growth in vivo.

(A) Flow cytometry analysis of CD163 and CD206 on THP-1 macrophages treated with different doses of α-lactose–supplemented GCM. (B) Flow cytometry analysis of CD86 on BMDMs or THP-1 macrophages treated with GCM, GCM + anti–Tim-3 antibody (1 μg/ml), or GCM + α-lactose (40 μM). (C to E) Survival curves of BALB/c-nu/nu mice bearing orthotopic U87 xenografts (C), C57BL/6J mice bearing orthotopic GL261 KO xenografts (D), and Wistar rats bearing orthotopic C6 xenografts (E) treated with α-lactose or anti–Tim-3 antibody. (F) Gal-9 knockdown prolonged the survival of BALB/c-nu/nu mice bearing orthotopic U87 xenografts. (G) Representative immunofluorescence staining and quantification of Gal-9/CD163 in orthotopic PTEN-Del U87 and PTEN-KO GL261 tumors treated with α-lactose or anti–Tim-3 antibody. Scale bar, 200 μm. ns, no significance. ***P < 0.001, one-way ANOVA. (H) Representative immunofluorescence staining and quantification of Gal-9/CD163 in orthotopic and subcutaneous U87-shC and U87-shGal-9 tumors. Scale bar, 200 μm. Tumors were harvested on day 22 after implantation. n = 3 biological replicates. ***P < 0.001, Student’s t test. In (C) to (F), *P < 0.05, **P < 0.01, and ***P < 0.001, log-rank test.

Fig. 7.. α-Lactose inhibits glioma angiogenesis by impairing TAM-derived VEGFA expression.

(A) Representative images of subcutaneous PTEN-KO GL261 tumors treated with or without α-lactose. (B) IHC showing CD31⁺ vessel density in intracranial PTEN-KO GL261 tumors treated with or without α-lactose. Scale bar, 200 μm. n = 3 biological replicates. (C and D) GSEA for VEGFA signatures and heatmap representation of 50 up-regulated VEGFA pathway–associated genes in healthy monocytes treated with or without GCM. (E) GSEA for two VEGFA signatures in M2 macrophage–positive versus M2 macrophage–negative correlated TCGA GBM samples. NES, normalized enrichment score. (F) VEGFA positively correlates with CD163 in the CGGA database. (G) qRT-PCR and (H) immunoblot validation of VEGFA expression in THP-1 macrophages and BMDMs treated with GCM or GCM + α-lactose. ***P < 0.001, Student’s t test. (I) Schematic model of the mechanism underlying how glioma cells with PTEN deficiency regulate macrophage polarization through Gal-9/Tim-3 signaling, which can be blocked by α-lactose or anti–Tim-3 antibody.