Glioblastomas exploit truncated O - linked glycans for local and distant immune modulation via the macrophage galactose-type lectin

Abstract

Glioblastoma is the most aggressive brain malignancy, for which immunotherapy has failed to prolong survival. Glioblastoma-associated immune infiltrates are dominated by tumor-associated macrophages and microglia (TAMs), which are key mediators of immune suppression and resistance to immunotherapy. We and others demonstrated aberrant expression of glycans in different cancer types. These tumor-associated glycans trigger inhibitory signaling in TAMs through glycan-binding receptors. We investigated the glioblastoma glycocalyx as a tumor-intrinsic immune suppressor. We detected increased expression of both tumor-associated truncated O-linked glycans and their receptor, macrophage galactose-type lectin (MGL), on CD163⁺ TAMs in glioblastoma patient-derived tumor tissues. In an immunocompetent orthotopic glioma mouse model overexpressing truncated O-linked glycans (MGL ligands), high-dimensional mass cytometry revealed a wide heterogeneity of infiltrating myeloid cells with increased infiltration of PD-L1⁺ TAMs as well as distant alterations in the bone marrow (BM). Our results demonstrate that glioblastomas exploit cell surface O-linked glycans for local and distant immune modulation.

Keywords: O-linked glycosylation; glioblastoma; immunosuppression; macrophage galactose lectin; macrophages.

Fig. 1.

Glioblastomas overexpress immature O-linked glycans. (A) Schematic representation of the O-glycosylation pathway. More than 20 different GALNTs have been characterized in humans that have overlapping activity but differ in peptide substrate specificities and expression pattern. GalNAc transferases catalyze the synthesis of GalNAcα1-O-serine/threonine (also known as the Tn antigen) as the first step in O-glycosylation (38). The Tn antigen is further elongated by the concerted action of several glycosyltransferases. (B) HPA binding to four glioblastoma cell lines (U87, U251, U373, and GBM8) measured by flow cytometry. (C) Heatmap of publicly available microarray data showing expression of genes encoding for glycosyltransferases and glycosidases in healthy brain tissue samples and 240 glioma WHO II (astrocytomas and oligodendrogliomas, n = 21), WHO III (astrocytomas and oligodendrogliomas, n = 60), and WHO IV (n = 159) samples. (D) Volcano plot showing 270 glycosylation-specific genes (SI Appendix, Table S1) with O-linked glycosylation related genes highlighted. C and D include a reanalysis of raw data obtained from Gravendeel et al. (35), including, for D, healthy brain tissue (n = 8), astrocytoma WHO II (n = 13), astrocytoma WHO III (n = 60), and glioblastoma (WHO IV, n = 159) samples. (E) Seven-micrometer cryosections of surgical epilepsy, LGG, and glioblastoma (GBM) samples, representative for five patients per condition, stained with HPA (green) and Hoechst as nuclear counterstain (blue). (F) Seven-micrometer cryosections of surgical epilepsy, LGG, and glioblastoma samples, representative for five patients per condition, stained with MGL-mouseFc (red) and Hoechst as nuclear counterstain (blue). (G) Human MGL-binding ELISA with lysates of glioblastoma (n = 12), LGG (n = 5), and epilepsy (n = 8) tissues, showing a significant difference (*P < 0.01, ***P = 0.005) between glioblastoma, and epilepsy samples (OD 450 nm).

Fig. 2.

TAMs in the glioblastoma microenvironment express MGL. (A) Human glioblastoma, LGG, and epilepsy tissues, representative for five patients per condition, stained for the MGL receptor (yellow) and nuclei (blue). (B) Human glioblastoma tissue stained for the MGL receptor (yellow), CD163 (red), and nuclei (blue). (C) MGL expression in glioblastoma, astrocytoma grades II and III, and control tissues. (D) CD163 expression in glioblastoma, astrocytoma grades II and III, and control tissues. (E) Scatterplot with expression of CD163 on the x axis and MGL on the y axis showing a moderate correlation in glioblastoma tissue (Pearson’s R = 0.29, P ≤ 0.0001). C−E include a reanalysis of raw data obtained from Gravendeel et al. (35), including, for C and D, healthy brain tissue (n = 8), astrocytoma WHO II (n = 13), astrocytoma WHO III (n = 60), and glioblastoma (WHO IV, n = 159) samples. (F) Kaplan−Meier curve showing survival benefit for patients with lower expression of MGL (n = 84) versus patients with higher expression of MGL (n = 85, P = 0.032, with median expression value of 3.25 as cutoff). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3.

High-dimensional mass cytometric analysis of a murine model for glioblastoma overexpressing immature O-linked glycans. (A) Overview level HSNE clustering of 12-mln cells pooled from brains, BM, and spleens of the 20 mice (10 MGL-L^HI and 10 WT tumors), showing cell density within different clusters, and expression of some key markers for cell lineage identification. (B) HSNE map, color-coded by cluster as identified in the first-level HSNE clustering. (C) Summary of cluster phenotypes. (D) HSNE maps selectively showing cells originating from brain, spleen, or BM. (E) Percentages of cells per cluster, relative to the total number of cells per organ (brain, spleen, or BM).

Fig. 4.

Glioblastoma-associated MGL-Ls attract CCR2⁺PD-L1⁺ macrophages. (A) HSNE map with the main clusters of interest highlighted. (B) HSNE map, colored by 16 subclusters as identified in the second-level HSNE subclustering of cluster F: tumor-infiltrating myeloid cells. (C) Density features depicting the local probability density of cells in cluster F. (D) Sample distribution. Each color represents a mouse, indicating a lack of sample bias in our data. (E) HSNE maps representing relative expression of the classifying myeloid markers (CD45, CD11b, Ly6C, Gr1, and CD11c). (F) HSNE maps representing relative expression of functional markers (Sca-1, CCR2, PDL-1, and PD-1). (G) Heatmap with relative ArcSinH(5) transformed expression of shown markers in the second-level HSNE of cluster F (Left) with a bar graph showing the percentages of total live cells in the brain per cluster (Right). (H) The three subclusters (F5, F7, and F16) with statistically significant differences in size between MGL-L^Hi and WT tumors. (I) Networks of subclusters from WT (Left) and MGL-L^Hi (Right) tumors with nodes visualizing all of the level-two cell subclusters in the experiment and edges representing correlation coefficients for relationships between subclusters. The size of the nodes represents the size of the population, the color intensity of the nodes represents the number of connections to other nodes, and the color intensity of the edges represents the correlation coefficient. The orange nodes highlight population F16, and the red nodes highlight the nearest neighbors of population F16 in both MGL-L^Hi- and WT tumors. ***P < 0.001.

Fig. 5.

Glioblastoma-associated MGL-Ls affect the myeloid composition of the BM. (A) HSNE map with the main clusters of interest highlighted. (B) HSNE map, colored by cluster as identified in the second-level HSNE clustering of cluster A: BM-derived myeloid cells. (C) Cell density. (D) Sample distribution. (E) HSNE maps representing relative expression of the classifying myeloid markers. (F) HSNE maps representing relative expression of functional markers. (G) Heatmap with relative Arcsinh transformed expression of shown markers in the second-level HSNE of population A (Left) with a bar graph showing the percentages of total live cells in the BM that the subclusters contain (Right). (H) The five subpopulations with statistically significant differences in relative frequency between mock transfected tumors and Cosmc KO tumors. (I) Networks of subclusters from WT (Left) and MGL-L^Hi (Right) tumors with nodes representing all of the subclusters in the experiment and edges representing correlation coefficients for relationships between the subclusters. The size of the nodes represents the size of the population, the color intensity of the nodes represents the degree (number of connections to other nodes), and the color intensity of the edges represents the correlation coefficient. The orange nodes highlight population A1, and the red nodes highlight the nearest neighbors of population A1 in both WT and Cosmc KO tumors. ***P < 0.001.