Fig. 1: Transcriptional programs and topology of HRS cells.

a Schematic of experimental design and analyses. Created in BioRender. Shanmugam, V. (2025) https://BioRender.com/nkuwxyx. b an overview of the single-nucleus RNA sequencing (snRNAseq) dataset. Uniform manifold approximation and projection (UMAP) embedding of snRNAseq profiles colored by cell type annotation (n = 308,403 nuclei from 15 subjects). c representative slide-seqV2 array of a Hodgkin lymphoma tumor with hematoxylin and eosin (H&E) stained section (top) and spatial plot of cell types colored by cell type (bottom), same color scheme as b. A total of 62 slideseqV2 arrays sampling different regions of samples from 19 subjects showed expected patterns of cell type distribution based on morphology. d identification of HRS cells by copy number variant (CNV) inference using inferCNV and EBV-specific read mapping. UMAP embedding showing CNV burden (quantified by CNV score) across cell types (n = 308,403 nuclei from 15 subjects). The top right inset shows the UMAP embedding of HRS cells identified by high CNV burden colored by EBV status determined by in situ hybridization (positive or negative). The lower right inset shows the same UMAP embedding with the expression levels of EBV-specific transcripts (n = 6720 nuclei from 10 patient samples). e normalized and scaled gene expression arranged by chromosome number showing CNVs in EBV- and EBV+ HRS cells (grouped by donor), relative to non-tumor cells (top) that show no significant evidence of CNVs. f Left: heatmap showing shared gene expression programs (normalized and scaled expression) in tumor cells across multiple donors identified by non-negative matrix factorization, grouped by EBV status and donors. Right: enrichment analysis results of curated gene sets (columns) in each program (rows). Pathway enrichment analysis was performed using a one-sided hypergeometric test and P values were adjusted for multiple comparisons using the Benjamini-Hochberg method. g spatial contour plot showing the clustered topology of HRS cells in tissue (representative array from donor H12-200). This analysis was repeated in 20 arrays with sufficient numbers of tumors cells and the majority of samples showed similar findings (Supplementary Data 4). For c and g, scale bars, 500 μm.