Additional file 1 of Spatial transcriptomic analysis of Sonic hedgehog medulloblastoma identifies that the loss of heterogeneity and promotion of differentiation underlies the response to CDK4/6 inhibition
Creators: name: Vo, Tuan
name: Balderson, Brad
name: Jones, Kahli
name: Ni, Guiyan
name: Crawford, Joanna
name: Millar, Amanda
name: Tolson, Elissa
name: Singleton, Matthew
name: Kojic, Marija
name: Robertson, Thomas
name: Walters, Shaun
name: Mulay, Onkar
name: Bhuva, Dharmesh D.
name: Davis, Melissa J.
name: Wainwright, Brandon J.
name: Nguyen, Quan
name: Genovesi, Laura A.
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Supplementary Figure S1AQ. Optimisation of the Spatial Transcriptomics technology for hybrid human-mouse tissue sections. This tiff file represents a graphical overview of the Visium Spatial Transcriptomics technology and the optimisation steps required for hybrid human-mouse tissue. Supplementary Figure S2. Structures of mouse histology can be identified within each sample using Visium spatial transcriptomics data, with high resolution and additional information about heterogeneity in gene expression present within one cluster. This tiff file represents a visual overview demonstrating how clustering analysis using gene expression data can define anatomically correct structures within the mouse cerebellum. Supplementary Figure S3. Spatial visualisation and quality control assessment of gene expression within Medulloblastoma Patient-derived orthotopic xenografts. This tiff file illustrates the total number of unique molecular identifiers, human genes and mouse genes per spot across the intact tissue section. Supplementary Figure S4. Diagnostic plots to assess technical variation and normalisation for gene expression analysis of Visium ST-seq data. This tiff file represents the normalisation and quality assessment of Visium ST-seq data across mouse, human and mixcompartments to identify differentially expressed genes in each compartment following Palbociclib treatment. The total number of unique molecular identifiers, human genes and mouse genes per spot across the intact tissue section. Supplementary Figure S5. Data-driven cell type detection for spatial transcriptomics spots were highly correlated to independent pathologist’s annotation based on histology. This tiff file illustrates independent pathologist annotationof Palbociclib A, B and control C overlaid with annotate cell types on the basis of Visium spot gene expression. Supplementary Figure S6. Overview of the reference single cell RNA-sequencingdata used for automated spot cell type identification. This tiff file contains two types of reference single cell RNA-sequencingdata used to annotate human/mix spots using human genes) and mouse/mix spots using mouse genes). Supplementary Figure S7. Definition of spots at the tumour-mouse interface. This tiff file represents the enrichment of cell types on the basis of gene expression for spots at the tumour-mouse interface of Palbociclib A, Palbociclib B, Control C, and Control. Supplementary Figure S8. Palbociclib-treatment induces the expression of genes associated with neuron differentiation in SHH MB. This tif file represents an immunofluorescence stain and immunoblot analysis of two markers for neuronal differentiation in Palbociclib treated and untreated control tumours, together with quantification. Supplementary Figure S9. Spatial response of cell proliferation to Palbociclib treatment in SHH-MB. This tif file represents an immunofluorescence stain and quantitative analysis for cell proliferation marker, Ki67, within the interface and bulk of Palbociclib treated tumours and untreated control PDOX. Supplementary Figure S10. Gene set activity scores of SHH-C and SHH-B groups. This tif file contains density plots of the normalised area of gene set activity scores for SHH-C and SHH-B in Palbociclib treated and control PDOX. Supplementary Figure S11. Per-spot enrichment analysis of differentially expressed neuron differentiation genes and neuronally differentiated SHH-C subpopulation correlates to regions independently annotated as ‘pale island’ by a pathologist. This tiff file illustrates a comparison between pathologist annotations from H&amp;E images, per-spot enrichment scores of differentially expressed genes annotated in the neuron differentiation signature and the SHH-C signaturein Palbociclib A, Control C and Control D. Supplementary Figure S12. Analyses of mouse-only tissue regions indicates that mouse astrocytes localise to the tumour-mouse interface. This tiff file indicates the enrichment of mouse astrocytes on the basis of cell annotation analyses and the expression of a common marker for astrocytes Gfap expression at the tumour-mouse interface in Palbociclib A and Control D. Supplementary Figure S13. Differential expression analysis of astrocytes at the interface. This tiff file includes the multiple differential expression analyses of spots annotated as containing mouse astrocytes in the interface compared to mouse only regions of Palbociclib treated tumours and untreated control PDOX. Supplementary Figure S14. Spatial interaction analvsis. Astrocvte and microalia interaction is enriched specificallv at the interface. This tiff file shows the result of unbiased cell-cell interaction analysis at the interface of Palbociclib-treated PDOX following a double layer permutation test for enrichment of ligand-receptor co-expression. This analysis tests for the expression of ligand and receptor pairs between neighbouring spots taking into account the cell type information. Supplementary Figure S15. The expression of cell proliferation marker MKI67 is localised tumour regions with expression of astrocyte marker, Gfap. This tif file contains the spatial transcriptomic analysis via smRNA-FISH/RNAscope to visualise the target RNA molecule expression of MKI67 and Gfap in Palbociclib treated and untreated control PDOX at a single cell resolution. DAPI images and H &amp;E images of the tumours are also shown for reference. Supplementary Figure S16. Quantitation of gene expression levels as measured by smRNA-FISH/RNAscope in untreated and Palbociclib-treated PDOX. This tif file contains the quantitative analysis of spatial transcriptomic analysis via smRNA-FISH/RNAscope to visualise the target RNA molecule expression of MKI67, Gfap, S100b and Cd68 in tumour regions across Palbociclib treated PDOX and untreated PDOX. Supplementary Figure S17. RNAScope analysis was used to investigate cell types comprising a multi-lateral network associated with tumour progression in untreated PDOX. This tif file contains the spatial transcriptomic analysis via smRNA-FISH/RNAscope to visualise the target RNA molecule expression of MKI67 and Gfap in untreated control PDOX at a single cell resolution.