Fig 2: Monocytes differentiate into Gpnmb-high macrophages or dendritic cells in the tumor microenvironment based on scRNA-seq analysis on GBM-mouse model.(a) Immune cells were re-clustered from GL261 mouse scRNA-seq and visualized in UMAP plot. Color encodes different cell clusters. (b) The percentage of each cell type in early and late stages of tumors. (c) Monocytes, dendritic cells, and macrophages were applied to trajectory analysis. Pseudo-temporal ordering of cells across cell types (top) and time points (bottom). (d) Heatmap plot showing the bifurcation of gene expression starting from monocytes. Arrows correspond to the direction of either dendritic cells or macrophages. Represented genes in this branched modeling are shown on the right. (e) The violin plot represents the gene expression levels in different transition states or (f) tumors collected at different time points. (g) Feature plot showing the expression of Gpnmb in early and late stages of tumors. (h) Left, immunofluorescence staining of GPNMB, Mac-3, and CD3 in GBM sections. Green color represents GPNMB, red color corresponds to Mac-3 and blue color indicates CD3 (Bar represents 200 µm). Right, quantitative analysis of the distance between GPNMB+ macrophages and CD3+ T cells (n=5 mice). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig 3: GPNMB derived from macrophages contribute to PN-MES cell state transition.Intercellular communications between stromal and tumor cells were inferred using Nichenet. (a) Ligands ranking from the sender cells (Macrophages, Microglia, Lymphocyte, EC and SMC/GBMEC) was predicted to communicate with target cells (tumor cells) based on interacting with MES-regulons. Red heatmaps indicate the Pearson correlation of prioritized ligands from high to low. The ligand expressions across cell types are shown on the left. Color encodes the average expression level across all cells (blue is low, red is high), while the dot size corresponds to the percentage of cells expressing the feature gene. The right heatmap indicates the potential interaction between ligands and targets/receptors. Color indicates the high or low interaction, respectively. (b) The relative expression of GPNMB in each cell type by analyzing the HPA dataset. (c) The highly correlated genes with GPNMB from the TCGA and CGGA datasets. (d) Relative GPNMB expression in different GBM subgroups. (e) Kaplan-Meier analysis of survival in patients with high- and low-expressed GPNMB. (f) Tumor-associated macrophages were freshly sorted from GL261-derived tumor cells, and subsequently co-cultured with proneural type of GBM spheroids in presence of neutralizing antibody of GPNMB. After constant stimulation, the expressions of EPAS1, FOSL2, and CEBPB were determined using RT-PCR. Statistical analysis by one-way ANOVA (n=4 independent biological replicates, mean ± SD). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)