Journal: Cell Reports Medicine
Article Title: Epstein-Barr virus orchestrates spatial reorganization and immunomodulation in the classic Hodgkin lymphoma tumor microenvironment
doi: 10.1016/j.xcrm.2026.102722
Figure Lengend Snippet: Spatial multi-omics framework reveals EBV-linked modulation of tumor proliferation and cytokine signatures within the cHL TME (A) Our customized multi-omics workflow to evaluate EBV-positive and EBV-negative cHL TME. (1) Biopsies from two cohorts of EBV-positive ( n patient = 22, n core = 22) and EBV-negative ( n patient = 24, n core = 22) patients were assembled into TMAs, where (2) one section was stained with anti-CD30 to visualize HRS cells through IHC (top) and the adjacent hybridized with a panel of >18,000 RNA probes targeting human and EBV genes and stained with three fluorescent antibodies for GeoMx (bottom). This allowed the (3) selection of 124 tumor-enriched ROIs ( n = 62 each) and (4) computational identification of single-cell marker expression within to enable the (5) annotation of tumor, T, and immune cell populations and (6) targeted whole-transcriptome capture within each cell region for (7) dissecting the key differences underlying distinct T cell populations and dysregulation between EBV-positive and EBV-negative cHL TME. (B) Representative IHC and immunofluorescent images of EBV-positive (left) and EBV-negative cHL (right) sections, with markers for HRS (CD30), CD8 T (CD3 + & CD4 − ), CD4 T (CD3 + & CD4 + ), memory T (CD45RO), and nuclei (Hematoxylin and SYTO13) shown in the smaller panels and the corresponding cell annotations shown in the larger panels. Scale bars: 100 μm. (C) Top: log2 fold enrichment plot of annotated cell regions between EBV-positive and EBV-negative cHL ROIs. Significance stars are only shown for significant comparisons. Two-sided Wilcoxon tests were conducted for all cell-type proportions; test results were BH adjusted with a 0.05 FDR. Unadjusted p value and BH corrected test results are in . Bottom: expression heatmap of the key genes associated with each annotated cell region. Expression heatmap of other T cell cytotoxic genes and EBV genes are respectively in A and B. (D) Volcano plots showing differences in gene expression between memory (CD45RO+) and naive T cells (CD45RO−) stratified by EBV status, with a few of the most differentially expressed genes indicated. CD8A , CD8B , and LAG3 transcripts are indicated by the green arrows. The volcano plot without EBV stratification is in C. (E) Orthogonal validation of G2M cell proliferation transcriptomic signatures between EBV-positive and EBV-negative tumor regions from GeoMx cohort (left) and Ki67+ HRS cell proportion from MIBI cohort (right). Two-sided Wilcoxon test was conducted for the G2M signature; none were performed for the Ki67+ cell proportion as it shows only two discrete values. (F) Volcano plot of EBV-positive- vs. EBV-negative-tumor-rich regions, with some of the highly differentially expressed host genes shown. CXCL9 and CCL17 are indicated by the green arrows. (G) Receptor-ligand analysis of chemokines that promote T cell recruitment. Significance stars are only shown for significant comparisons. p values were generated from one-sided permutation tests, with the alternative hypothesis that the distribution of a given interaction is greater than the null distribution. Significant stars: ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001.
Article Snippet: After washing twice in washing buffer for 5 min each, the DAB peroxidase substrate (Vector Laboratories SK-4100) was introduced, and brown coloration was allowed to develop over 2.5 min. Tissue slides were briefly rinsed in tap water and counterstained with hematoxylin (Vector Laboratories H3404-100) for 2 min, and blue coloration was allowed to develop by rinsing in tap water for five times at 3 min each.
Techniques: Biomarker Discovery, Staining, Selection, Single Cell, Marker, Expressing, Gene Expression, Generated
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