Fig. 1

EBNA1 modulates the tumor microenvironment by suppressing inflammatory responses. a EBNA1 protein was overexpressed in both melanoma B16 and NPC HK1 cells. NC cells represent control tumor cells, and OE EBNA1 cells represent EBNA1-overexpressing tumor cells. The data are representative of three independent experiments. b Representative images of subcutaneous tumors in severe immunodeficient NCG mice, including HK1-NC tumors (left) and HK1-OE EBNA1 tumors (right), with corresponding tumor volumes and weights. The mouse diagram was created via FigDraw. The data are presented as the means ± s.e.m.s (n = 6 mice in each group). c Representative images of tumors from C57BL/6 mice injected with either NC or OE EBNA1 B16 cells (left), the corresponding tumor volume (middle), and the tumor weight (right) are shown. The mouse diagram was created via FigDraw. The data are shown as the means ± s.e.m.s n = 6 mice per group. d Experimental design for single-cell analysis or FACS validation of CD45⁺ cells sorted from tumors with or without EBNA1 expression. e, f t-distributed stochastic neighbor embedding (t-SNE) plot (c) and density plot (d) of 18,886 RNA sequencing (RNA-seq) single CD45⁺ cells from B16-OE EBNA1 and B16-NC tumors. M1, M1 macrophages; M2, M2 macrophages; cDCs, conventional dendritic cells; MoDCs, monocyte-derived dendritic cells; pDCs, plasmacytoid dendritic cells. g Single-cell gene set enrichment scores of the IFNγ response signature in individual immune subsets from EBNA1-overexpressing and control tumors (P-values were calculated via the Kolmogorov‒Smirnov test). n = 18,886 cells. h Gene set enrichment analysis (GSEA) was performed on immune cells from EBNA1-overexpressing tumors and control tumors to analyze the interferon response signature. i Protein levels of IFNβ and IFNγ in the TME of EBNA1 overexpressing and control B16 tumors. n = 6 in each group. j Flow cytometry analysis of the proportion of CD8⁺ T cells in the tumor microenvironment using the tumor-derived cells shown in (d). Each group consisted of 6 samples (n = 6). k Flow cytometry analysis of the proportion of M2 macrophages in the tumor microenvironment using the tumor-derived cells shown in (d). There were 6 samples per group (n = 6). l Immunohistochemistry (left) and quantification (right) of CD8⁺ T cells in NPC patient samples (black, n = 40) or NPE samples (red, n = 45). b Growth curves, c Growth curves, two-way ANOVA. b Tumor weight. c Tumor weight. i‒l Two-tailed unpaired t-test; *P < 0.05; **P < 0.01; ****P < 0.001; ns, not significant