Single-cell RNA sequencing analysis of vestibular schwannoma reveals functionally distinct macrophage subsets

Abstract

Background: Vestibular schwannomas (VSs) remain a challenge due to their anatomical location and propensity to growth. Macrophages are present in VS but their roles in VS pathogenesis remains unknown.

Objectives: The objective was to assess phenotypic and functional profile of macrophages in VS with single-cell RNA sequencing (scRNAseq).

Methods: scRNAseq was carried out in three VS samples to examine characteristics of macrophages in the tumour. RT-qPCR was carried out on 10 VS samples for CD14, CD68 and CD163 and a panel of macrophage-associated molecules.

Results: scRNAseq revealed macrophages to be a major constituent of VS microenvironment with three distinct subclusters based on gene expression. The subclusters were also defined by expression of CD163, CD68 and IL-1β. AREG and PLAUR were expressed in the CD68+CD163+IL-1β+ subcluster, PLCG2 and NCKAP5 were expressed in CD68+CD163+IL-1β- subcluster and AUTS2 and SPP1 were expressed in the CD68+CD163-IL-1β+ subcluster. RT-qPCR showed expression of several macrophage markers in VS of which CD14, ALOX15, Interleukin-1β, INHBA and Colony Stimulating Factor-1R were found to have a high correlation with tumour volume.

Conclusions: Macrophages form an important component of VS stroma. scRNAseq reveals three distinct subsets of macrophages in the VS tissue which may have differing roles in the pathogenesis of VS.

Fig. 1. Single-cell RNA analysis of cellular subsets in vestibular schwannoma (VS).

a The merged Uniform Manifold Approximation and Projection (UMAP) plot of 8808 VS cells from three patients (AN014, AN017 and AN018) showing the presence of 7 subclusters of cells based on expression of specific markers. b Expression of marker genes in the seven subclusters on scRNA analysis CD68, SOX2, CD3, PMP2, S100B, PECAM1, MK167, Decorin (DCN). c UMAP single-cell RNA analysis of the three patients presented individually. d Stacked bar chart of seven major cell-type composition of each patient. e Pie chart showing the composition of the VS tumour on single-cell analysis.

Fig. 2. Single-cell RNA analysis of macrophage subsets in vestibular schwannoma (VS).

The single-cell RNA analysis of the macrophage subset in three VS tissue samples from different patients. a Expression of CD68 in the macrophage subset on single-cell RNA analysis. b Integrated dataset showing the subclusters of macrophages based on contribution from each donor. c The integrated dataset showing four subclusters (red, dark blue, green and purple) of macrophages based on gene expression. d The analysis in the four subsets showing the ten highly expressed genes as a heat map. Cluster 0, Cluster 1 and Cluster 2 represent macrophage subsets. Cluster 3 are contaminating T cells. e Pathway analysis of the macrophage subsets on scRNA sequencing is shown (Go Terms). f DoroThea plot of the macrophage subclusters 0, 1 and 2 showing the expression of transcriptional factors in the three subsets.

Fig. 3. Expression of CD163 and IL-1β in macrophage subsets in vestibular schwannoma (VS) on scRNA analysis.

Single-cell RNA analysis of the macrophage subsets in three vestibular schwannoma tissue samples from different patients. a Expression of CD163, CD14 and CSFR1 in the macrophage subsets. b Expression of INHB-A, ALOX-15 and IL-1β in the macrophage subsets. c Expression of VEGF-A, TNF-α and IL-10 in the macrophage subsets. d Expression of TMEM119, CCL3 and CCL4 in the macrophage subsets.

Fig. 4. Expression of CD68, CD163 and IL-1β in vestibular schwannoma (VS) tissue on immunohistochemistry.

Immunohistochemistry images of VS tissue (representative of n = 8 VS patient samples). a IL-1β is in brown, nuclei are blue (middle and right panels), left-hand side tissue section depicts control staining. b CD163 is in pink and IL-1β is in brown. Middle and right panels are enlarged images taken from the areas indicated by the arrow/arrowhead on the left-hand side tissue section showing CD163/IL-1β double co-expressing macrophage (arrow) and IL-1β expressing but CD163 negative cell (arrowhead), respectively. c CD68 is in pink and IL-1β is in brown. Right side panel is image is taken from the area indicated by the black box on the left-hand side tissue section showing CD68 and IL-1β expressing macrophages.