Tumor heterogeneity and tumor-microglia interactions in primary and recurrent IDH1-mutant gliomas

Abstract

The isocitrate dehydrogenase (IDH) gene is recurrently mutated in adult diffuse gliomas. IDH-mutant gliomas are categorized into oligodendrogliomas and astrocytomas, each with unique pathological features. Here, we use single-nucleus RNA and ATAC sequencing to compare the molecular heterogeneity of these glioma subtypes. In addition to astrocyte-like, oligodendrocyte progenitor-like, and cycling tumor subpopulations, a tumor population enriched for ribosomal genes and translation elongation factors is primarily present in oligodendrogliomas. Longitudinal analysis of astrocytomas indicates that the proportion of tumor subpopulations remains stable in recurrent tumors. Analysis of tumor-associated microglia/macrophages (TAMs) reveals significant differences between oligodendrogliomas, with astrocytomas harboring inflammatory TAMs expressing phosphorylated STAT1, as confirmed by immunohistochemistry. Furthermore, inferred receptor-ligand interactions between tumor subpopulations and TAMs may contribute to TAM state diversity. Overall, our study sheds light on distinct tumor populations, TAM heterogeneity, TAM-tumor interactions in IDH-mutant glioma subtypes, and the relative stability of tumor subpopulations in recurrent astrocytomas.

Keywords: IDH mutation; astrocytoma; bone-marrow-derived macrophages; glioma; microglia; oligodendroglioma; recurrent glioma; snATAC-seq; snRNA-seq; tumor microenvironment.

Graphical abstract

Figure 1

snRNA-seq identifies gene expression programs driving cell states in IDH-mutant gliomas (A) Clinical and molecular characteristics of the IDH-mutant glioma cohort for single-nuclei sequencing. (B and C) UMAP representation and initial cluster assignments for integrated snRNA-seq samples of oligodendrogliomas (n = 8) (B) and astrocytomas (n = 6) (C). Nuclei labeled as “excluded” in (B) are omitted from downstream analyses due to technical reasons (tumor cells clustering with microglia and originating from a single patient). UMAP1, x axis; UMAP2, y axis. Colors indicate cell-type assignments. (D and E) Pearson’s correlation scores for individual NMF programs in oligodendrogliomas (n = 8) (D) and astrocytomas (n = 6) (E). Metaprograms were identified by hierarchical clustering of individual NMF programs. (F) Dot plot displaying five marker genes for each tumor population. (G and H) UMAP embedding of oligodendrogliomas (G) and astrocytomas (H), colored by NMF metaprograms, gradient cells, and TME (dark gray). (I) Bar plot showing tumor population proportions across individual samples, grouped by subtype and grade. Bars are arranged by descending RE proportion. (J) Representative IHC staining for H&E and two RE population markers, EEF2 and EEF1A1, for oligodendroglioma and astrocytoma samples, separated by grade. Oligodendroglioma (OD) n = 22 (11 grade 2, 11 grade 3); astrocytoma (AS) n = 15 (10 grade 2, 5 grade 3). Scale bars represent 50 μm. (K) Spatial distribution of EEF2 and EEF1A1 (40×). Scale bars represent 50 μm. (L) Plots showing semi-quantitative histological scores (0 = 0%, 1 = 0%–5%, 2 = 6%–29%, 3 = 30%–69%, and 4 >70% positive staining) for EEF2 (top) and EEF1A1 (bottom). Top: a qualitative increase in EEF2 staining in grade 3 compared with grade 2 AS tumors. Bottom: a qualitative increase in EEF1A1 staining in grade 2 OD tumors compared with grade 2 AS tumors. Wilcoxon rank-sum test was performed to test for significance, ∗p < 0.05.

Figure 2

Integrated snRNA-seq and snATAC-seq indicates a stem-like signature in the RE tumor population (A) Diffusion map visualization of tumor cells in OD and AS tumors, based on publicly available astro-like, oligo-like, and stemness program markers. Right: scaled and centered enrichment scores for NMF metaprograms. Cells are ordered along diffusion components 1 (DC_1) and 2 (DC_2); gradient cells were excluded for clarity. (B) Scaled and centered pathway activity scores (normalized weighted mean from snRNA-seq) for OD and AS tumors. (C) Scaled and centered regulon activity scores (normalized weighted mean from snRNA-seq) for OD and AS tumors. (D and E) UMAP embedding of OD (D) and AS (E) snATAC-seq data (OD n = 4, AS n = 5) colored by labels transferred from snRNA-seq datasets. UMAP1, x axis; UMAP2, y axis. (F) Bar plot showing proportion of cell types in snATAC-seq data. (G) Pearson correlation of highly variable peaks from snATAC-seq data in OD and AS tumors. (H and I) Heatmap showing the top significantly enriched transcription factor motifs in OD (H) and AS tumors (I). Scores are scaled and centered for clarity.

Figure 3

Integrated TAM states highlight differences between AS and OD tumors (A) UMAP embedding of integrated microglia from snRNA-seq data of primary OD and AS tumors, colored by assigned TAM subpopulations. UMAP1, x axis; UMAP2, y axis. (B) Bar plots indicating TAM subpopulation proportions, separated by tumor subtype and grade. (C) Boxplots showing TAM subpopulation proportions by tumor type. OD, oligodendroglioma; AS, astrocytoma. Wilcoxon rank-sum test was performed to test for significance. (D) Representative IHC staining for TAM markers IBA1, CD74, and CD163 and double staining for p-STAT1 and IBA1 (60×) in OD and AS tumors. Images are captured at either 40× (scale bar, 50 μm) or 60× original magnification (scale bar, 30 μm) (E) Plots showing semi-quantitative histological scores (0 = 0%, 1 = 0%–5%, 2 = 6%–29%, 3 = 30%–69%, 4 >70% positive staining) for p-STAT1⁺IBA1⁺ TAMs in OD and AS tumors, separated by grade (n = 3 per group). (F and G) Select statistically significant receptor-ligand interactions between OPC-like and astro-like tumor populations (source) and the TAM subpopulations (target) (F) and TAM subpopulations (source) and OPC-like and astro-like tumor cells (target) (G). Dot size represents significance (adjusted p values); dot color reflects expression magnitude (means of average expression level of the interacting pair of genes).

Figure 4

snRNA-seq of paired primary and recurrent AS cohorts indicates shifts in tumor populations associated with grade (A) Clinical and molecular characteristics of 6 paired primary and recurrent IDH-mutant AS cohorts for snRNA-seq. (B) UMAP embedding of integrated snRNA-seq data from paired AS tumors, colored by assigned cell types. UMAP1, x axis; UMAP2, y axis. (C) Bar plots showing tumor population proportions, separated by pairs, relapse status, and grade. (D) Representative IHC staining for RE markers EEF2 and EEF1A1 in paired primary (grade 2) and relapse (grade 3) AS tumors. Images are captured at 40x original magnification (scale bar, 50 μm) (E) Heatmap showing semi-quantitative EEF1A1 histological scores (0 = 0%, 1 = 0%–5%, 2 = 6%–29%, 3 = 30%–69%, 4 >70% positive staining) for 6 pairs of primary and recurrent OD and AS tumors. Wilcoxon signed-rank test for paired samples was performed to test for significance, ∗p < 0.05. (F) UMAP embedding of integrated microglia population of paired primary and recurrent AS cohorts, colored by assigned TAM subpopulations. UMAP1, x axis; UMAP2, y axis. (G) Bar plots showing TAM subpopulation proportions separated by pairs, relapse status, and grade. (H) Boxplots showing TAM subpopulation proportions separated by relapse status. P, primary; R, relapse.