Fig. 3: Spatial organization of the immune microenvironment around Hodgkin Reed Sternberg cells.

a Mean enrichment ratios of immune cell types (colored by cell type) aggregated across the entire multi-sample, multi-replicate slideseqV2 dataset (n = 38, independent arrays sampling different tumor regions from 12 patients). Statistical significance was assessed by calculating z-scores for log₂(mean enrichment ratios) relative to zero and deriving two-sided P values from the standard normal distribution. The size of the points indicates the P-value; mean ± s.e.m. is depicted. b representative array showing spatial enrichment of CD4+ T cells and depletion of plasma cells around clustered HRS cells. The images are representative of 38 independent arrays sampling different tumor regions from 12 patients. Scale bar: 500 μm. c volcano plot showing cell-type specific differentially expressed genes in relation to HRS cell proximal/dense regions. A positive log₂(fold change) (right of volcano plot) indicates genes that show higher expression in HRS cell proximal/dense regions. Fold changes and q-values are calculated using the C-SIDE framework (see Methods). q-values denote p-values (two-sided) adjusted for multiple comparisons using the Benjamini–Hochberg false discovery rate (FDR) method. Results are aggregated across the entire multi-sample, multi-replicate slide-seqV2 dataset (n = 38, independent arrays sampling different tumor regions from 12 patients). d CosMx analysis showing enrichment of IL10+ CD4+ T cells within HRS cell dense/proximal regions. This image is representative of multiple fields of view sampling 4 cHL samples. Scale bar, 100 μm. e schematic representation of the structured model where immune cells show a non-random spatial distribution around clustered tumor cells in contrast to the null hypothesis random spatial model where immune cells are randomly distributed. Created in BioRender. Shanmugam, V. (2025) https://BioRender.com/1uaubi2. f schematic overview of the approach for systematic discovery and prioritization of spatially-informed tumor-immune ligand-receptor interactions within the HRS cell niche. Created in BioRender. Shanmugam, V. (2025) https://BioRender.com/0hekpqv. g scatterplot of tumor-targeted ligand-receptor interactions based on spatial proximity (spatial association metric, see methods) and normalized ligand/receptor expression (sca-LRscore metric). Interactions that show high ligand/receptor expression and spatial proximity are called out and shown on the right as a bubble plot. h RNA fluorescence in-situ hybridization analysis of IL13 expression (yellow) in HRS cells and the microenvironment. The red TNFRSF8 (encoding CD30) probe highlights HRS cells. Scale bar; 20 µm. The data are representative of independent experiments on three different patient samples. i schematic diagram of systematically prioritized paracrine tumor-microenvironment interactions in Hodgkin lymphoma, including putative growth factors and immunosuppressive interactions.