Martha A. Cady

Supplementary Fig. 12. Single-cell RNA sequencing of human and mouse cells from meningioma xenografts. A, Single-cell counts across experimental conditions, biological replicates (samples), and UMAP clusters from CH-157MN meningioma xenograft human cells analyzed using single-cell RNA sequencing. B, Single-cell counts across experimental conditions, biological replicates, and phases of the cell cycle from meningioma xenograft human cells analyzed using single-cell RNA sequencing. Colors and rows (samples) as in A. C, Single-cell counts across experimental conditions, biological replicates, and UMAP clusters from meningioma xenograft mouse cells analyzed using single-cell RNA sequencing. D, Stacked bar plots showing the distribution of meningioma xenograft human single-cell RNA sequencing clusters across biological replicates. Colors as in A. E, Stacked bar plots showing the distribution of meningioma xenograft mouse single-cell RNA sequencing clusters across biological replicates. Colors as in C.

Supplementary Fig. 6. WHO grade 3 meningioma immunofluorescence microscopy for NOTCH3 and the mural cell marker SMA. NOTCH3 is expressed in and out of the perivascular niche, and colocalizes with mural cells in the perivascular niche, in meningiomas with WHO grade 3 histology. SF numbers indicate individual meningiomas. Representative of n=10 WHO grade 3 meningiomas. WHO grade defined using histological criteria. Scale bars, 10µm.

Supplementary Fig. 8. WHO grade 2 meningioma immunofluorescence microscopy for NOTCH3 and the endothelial cell marker VWF. NOTCH3 expression is primarily restricted to the perivascular niche adjacent to endothelial cells in meningiomas with WHO grade 2 histology. SF number indicates meningioma identifier. Representative of n=10 WHO grade 2 meningiomas. WHO grade defined using histological criteria. Scale bar, 10µm.

Supplementary Fig. 7. WHO grade 1 meningioma immunofluorescence microscopy for NOTCH3 and the endothelial cell marker VWF. NOTCH3 expression is restricted to the perivascular niche adjacent to endothelial cells in meningiomas with WHO grade 1 histology. SF numbers indicate individual meningiomas. Representative of n=10 WHO grade 1 meningiomas. WHO grade defined using histological criteria. Scale bars, 10µm.

Supplementary Fig. 4. WHO grade 1 meningioma immunofluorescence microscopy for NOTCH3 and the mural cell marker SMA. NOTCH3 expression is restricted to the perivascular niche and colocalizes with mural cells in meningiomas with WHO grade 1 histology. SF numbers indicate individual meningiomas. WHO grade defined using histological criteria. Representative of n=10 WHO grade 1 meningiomas. Scale bars, 10µm.

Supplementary Fig. 1. Single-cell RNA sequencing of human meningiomas with loss of chromosome 22q. A, Single-cell counts across UMAP clusters from human meningioma samples with loss of chromosome 22q analyzed using single-cell RNA sequencing. B, UMAP showing single-cell RNA sequencing of human meningioma samples shaded by sample of origin. C, Stacked bar plots showing the distribution of chromosome 22q loss (red) across single-cell RNA sequencing clusters from human meningioma samples. D, UMAP and stacked bar plot showing single-cell RNA sequencing cell cycle analysis across human meningioma samples. E-G, Heatmap and feature plots showing differentially expressed genes across UMAP clusters from human meningioma samples analyzed using single-cell RNA sequencing. Colors as in A. See also Supplementary Table 1. G, Feature plots showing expression of meningioma (SSTR2, PTGDS) or cancer stem-cell markers (NOTCH3, THY1) across UMAP clusters from human meningioma samples analyzed using single-cell RNA sequencing.

Supplementary Fig. 16. NOTCH3 inhibition blocks meningioma xenograft growth without causing toxicity. A, A NOTCH1 negative regulatory region neutralizing antibody (αNRR1), but not αNRR3, causes diarrhea and rash resulting in weight loss in mice harboring meningioma xenografts. Antibodies were delivered using biweekly IP injection. Student’s t tests. B, The -secretase inhibitor LY-411575 (GSI) delivered using daily IP injection causes diarrhea and rash resulting in weight loss in mice harboring meningioma xenografts. Student’s t tests. C, LY-411575 attenuates the growth of CH-175MN (left) or IOMM-Lee (right) meningioma xenografts in mice. Student’s t tests. Lines represent means and error bars represent standard error of means.

Supplementary Fig. 8. WHO grade 2 meningioma immunofluorescence microscopy for NOTCH3 and the endothelial cell marker VWF. NOTCH3 expression is primarily restricted to the perivascular niche adjacent to endothelial cells in meningiomas with WHO grade 2 histology. SF number indicates meningioma identifier. Representative of n=10 WHO grade 2 meningiomas. WHO grade defined using histological criteria. Scale bar, 10µm.

Supplementary Fig. 14. Meningioma invasion of Virchow-Robin spaces. A, H&E low magnification (top) and high magnification (box, bottom) images of human meningioma invasion into perivascular fluid-filled cavities surrounding perforating vasculature of the brain. Densely cellular meningioma is shown at the top of each image, and islands of perivascular tumor within the brain parenchyma (dashed lines, bottom) are shown at the bottom of each image. Scale bars, 100µm. B, Low magnification (top) and high magnification (box, bottom) images of IHC for the brain parenchyma marker GFAP validates invasion of unlabeled meningioma cells into Virchow-Robin spaces without direct invasion of the brain parenchyma itself. C, IHC for the mural cell marker SMA (top) or the meningioma cell marker SSTR2A (bottom) validates meningioma invasion into Virchow-Robin spaces.

Supplementary Fig. 2. Single-cell RNA sequencing of dog meningiomas. A, Single-cell counts across UMAP clusters from dog meningioma samples analyzed using single-cell RNA sequencing. B, UMAP showing single-cell RNA sequencing of dog meningioma samples shaded by sample of origin. C, UMAP showing single-cell RNA sequencing cell cycle analysis across dog meningioma samples. D-E, Heatmapt and feature plots showing differentially expressed genes across UMAP clusters from dog meningioma samples analyzed using single-cell RNA sequencing. Colors as in A. F, Magnetic resonance imaging (MRI, left) or H&E images (right) of spontaneous dog meningiomas. Scale bars, 100µm. See also Supplementary Table 2.