Single-cell RNA-seq of cells from tumor center, periphery and blood.

(A) Experimental workflow for single-cell analysis of cells isolated from tumor center, periphery and peripheral blood mononuclear cells (PBMC), including fluorescent-activated cell sorting and 3’-scRNA-seq. (B) Axial T1 with contrast (left) and T2 (right) MRI brain in a patient with a left temporal GBM. Fresh tumor biopsies were taken according to neuronavigation (green cross). The tumor center was defined as contrast enhancing, whereas the tumor periphery was defined as T2 hyperintense. (C) Nuclear DAPI staining of resected tissue specimens. 40x magnification (scale bar = 20 μm). n = 3 patients, 4 field of view (FOV) per patient. Statistics: ***p < 0.001, two-tailed Mann Whitney U test (Figure 1 – source data 1).

Single-cell RNA-seq analysis identifies main immune cell populations.

(A) Dimensionally reduced tSNE projection of the scRNAseq data showing the annotated cell types. (B) Heatmap displaying centered and scaled normalized average expression values of characteristic cell-type specific genes used to annotate clusters. Columns are ordered by site and cell type, and rows show centered and scaled expression values, hierarchically clustered. Heatmap displaying genes whose expression is most specific to each cell type is shown in Figure 2 – figure supplement 2. (C) Principal component analysis of pseudo-bulk scRNAseq samples aggregated by patient and cell type. Symbols represent individual patients and cell lineage is displayed by different colors. (D) Relative frequencies of immune populations among leukocytes between tumor center and periphery. Symbols represent individual patients and paired samples are indicated by connecting lines. P-values were calculated using diffcyt-DA-voom method (Figure 2 – source data 1). (E-G) Differential abundance testing of the tumor innate immune compartment (E) using the miloR package which tests the abundance of each neighborhood of cells separately between tumor center und periphery (F, G).

MG display regionally resolved transcriptional profiles that differ from those of DCs and MdMs.

(A) Microglia cluster highlighted on tSNE map and scatterplots showing differentially expressed genes (FDR<5%, indicated by blue and yellow) in Microglia (MG) cells from tumor periphery versus center. Volcano plot showing p value versus fold-change (left) and MA plot showing fold-change versus mean expression (right). For a complete list of differentially expressed genes per cell cluster between tumor periphery and center, please refer to Supplementary File 4. (B) Heatmap representation of Gene set enrichment analysis (GSEA) results between peripheral and center microglia using Gene Ontology (GO) collection (Biological Processes). The fraction of overlap between gene sets is calculated as Jaccard coefficient of overlap between the gene sets. (C, D) Heatmap representation of GSEA of DCs (C) and monocytederived macrophages (MdMs) (D) from tumor periphery versus tumor center using Hallmark collection of major biological categories. (E) Unsupervised hierarchical sub-clustering of the MG population revealed two transcriptionally distinct subsets of MG, termed MG_1 and MG_2, displayed on the tSNE map. (F) Heatmap displaying the cluster-specific genes identifying MG_1 subcluster. Columns are ordered by site and cell type, and rows show centered and scaled normalized average expression values, hierarchically clustered. A complete list of cluster specific genes for MG_1 and MG_2 subcluster is provided in Supplementary File 5. (G) Heatmap representation of GSEA between MG_1 and MG_2 subclusters using Hallmark collection of major biological categories. (H) Heatmap displaying previously described reactivity markers of MG. Columns are ordered by site and cell type, and rows show centered and scaled normalized average expression values, hierarchically clustered.

The peripheral cytotoxic cell compartment exhibits an impaired activation signature.

(A, B) Volcano plots showing differentially expressed genes (FDR corrected p value < 0.05, indicated by blue and yellow) in CD8+ T cells (A) and NK cells (B) from tumor periphery versus tumor center. Colored rings mark genes belonging to selected GSEA Hallmark or Gene Ontology (GO) pathways as indicated. For a complete list of differentially expressed genes per cell cluster between tumor periphery and center, please refer to Supplementary File 4.

CX3CR1 labels a specific CD8+ T cell population in the circulation of grade 4 glioma patients.

(A) CD8+ T cell cluster highlighted on tSNE map (left). CD8+ T cell cluster colored by site of origin (right). (B) Volcano plot showing differentially expressed genes (FDR corrected p value < 0.05, indicated by blue and green) in CD8+ T cells from tumor-periphery versus PBMC. For a complete list of differentially expressed genes per cell cluster between tumor periphery and PBMC, please refer to Supplementary File 6. (C) Frequency of CX3CR1+ CD8+ T cells among all CD8+ T cells in flow cytometry data (Figure 5 – source data 1). (D) Unsupervised hierarchical sub-clustering of CD8+ T cells from PBMC and Periphery revealed two transcriptionally distinct subsets of PBMC CD8+ T cells, displayed on the tSNE map. (E) Expression of CX3CR1 overlaid on tSNE CD8+ T cell cluster. (F) Expression of genes associated with effector memory phenotype overlaid on tSNE CD8+ T cell cluster. Displayed genes are significantly, differentially expressed genes (DEGs) between tumor periphery and PBMC, as identified by differential gene expression analysis shown in panel (B). (G) Expression of selected genes associated with naive phenotype overlaid on tSNE CD8+ T cell cluster (H) Gating procedure applied to identify CD3+ CD8+ naïve, T effector cells (Teff), effector memory (Tem), peripheral memory (Tpm) and central memory (Tcm), eluted from PBMCs. (I) Expression of CX3CR1 in PBMC CD8+ T cell subpopulations identified in (H) (Figure 5 – source data 2). n = 6 donors (C), n = 11 donors (I). Statistics: Wilcoxon matched-pairs signed rank test (C); repeated measures oneway ANOVA with post-hoc Šidák’s correction for multiple comparisons (I). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, no brackets indicate no significant difference.

CD8+ T cells in the tumor periphery share features with tissue-resident memory T cells (Trm)

(A) Representative dot plot of tumor-periphery CD8+ T cells stained for CD45RA and CD45RO. (B) Quantification of tumor-periphery CD8+ T cells expressing CD45RA or CD45RO (Figure 6 – source data 1). (C) Expression of genes associated with tissue-resident memory (Trm) phenotype overlaid on tSNE CD8+ T cell cluster. (D) Average expression levels of selected Trm markers between CD8+ T cells from PBMC versus tumor-periphery. Significance testing based on differential gene expression analysis shown in panel (Fig. 5B) (E) Representative dot plots of CD69 and CD103 co-expression in CD8+ T cells from PBMC and tumor-periphery. (F) Quantification of CD69 and CD103 co-expression revealed CD69- CD103- in PBMC and CD69+ CD103- and CD69+ CD103+ in tumor-periphery as the dominant phenotypes (Figure 6 – source data 2). (G) Expression of selected markers associated with T cell exhaustion/dysfunction, shown as boxplots between CD8+ T cell from PBMC and tumorperiphery and overlaid on tSNE CD8+ T cell cluster. n = 6 donors (B, F). Statistics: Wilcoxon matched-pairs signed rank test (B); repeated measures one-way ANOVA with post-hoc Šidák’s correction for multiple comparisons (F). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, no brackets indicate no significant difference.

Cell-cell communication analysis using CellChat reveals critical role for SPP1-mediated crosstalk in tumor periphery.

(A) Chord diagram showing significant interactions from microglia to lymphocyte cell clusters. The inner bar colors represent the targets that receive signal from the corresponding outer bar. The inner bar size is proportional to the signal strength received by the targets. Chords indicate ligand-receptor pairs mediating interaction between two cell clusters, size of chords is proportional to signal strength of the given ligand-receptor pair. (B) Comparison of incoming and outgoing interaction strength allows identification of main senders and receivers. (C) Violin plots showing the expression distribution of signaling genes involved in the inferred SPP1 signaling network.