Extended Data Fig. 9: Tumour EVPs promote TNF secretion from Kupffer cells.

a, Representative IF image of the primary Kupffer cells isolated from naïve mice. F4/80 in red and DAPI in blue. Shown is a representative image from three independent experiments. Scale bar, 20 µm. b, ELISA analysis of IL-6 secretion from naïve mice-derived Kupffer cells after treatment with LPS (1 µg ml⁻¹). n = 3 independent experiments per group. c,d, Representative cytokine array blots and associated quantification charts for the cytokines and chemokines in the whole cell lysates of Kupffer cells isolated from B16F10-Tb mice (c) and K7M2-Tb mice (d), and their respective PBS-injected controls. e,f, Representative cytokine array blots and associated quantification charts for the cytokines and chemokines in the conditioned media of Kupffer cells isolated from naïve mice and educated with 10 µg ml⁻¹ of B16F10-CL-EVPs (e) or K7M2-CL-EVPs (f) in vitro for 3 days, and their respective PBS-educated controls. g, TNF ELISA on the conditioned medium of Kupffer cells educated with 10 µg ml⁻¹ of B16F10-CL-EVP- (left), or K7M2-CL-EVP- (right), and their respective PBS controls for 3 days. n = 3 independent experiments per group. h, RT-qPCR analysis of Tnf expression in Kupffer cells isolated from B16F10-Tb (left), or K7M2-Tb (right) mice, and their respective PBS-injected controls. n = 4 mice per group. i, RT-qPCR analysis of Tnf expression in Kupffer cells treated with control PBS, EVPs derived from B16F10 cells or B16F10 tumour explants (left), or EVPs derived from K7M2 cells or K7M2 tumour explants (right) for 4 h. n = 3 independent experiments per group. j, TNF ELISA on EVP-depleted conditioned media from B16F10 and K7M2 cells. n = 2 independent experiments. ND, not detected. CM, conditioned medium. k, GSEA of gene expression profiles, ranked based on the sign of log₂FC∗(-log₁₀P value), in hepatocytes 24 h post treatment with recombinant mouse TNF protein (25 ng ml⁻¹), compared to PBS control, using Gene Ontology gene sets. Shown are downregulated lipid catabolism-associated gene sets. NES, normalized enrichment score. NOM p-value, nominal p-value. l, Schematic Illustration of anti-TNF antibody (200 µg per mouse) or IgG1 isotype control (200 µg per mouse) treatment of tumour-bearing mice or tumour EVP-educated mice. I.P., intraperitoneal. m, Weights of tumours from mice inoculated with B16F10 (left) or K7M2 (right) cells and treated with anti-TNF antibody or IgG1 isotype control as shown in (l). n = 5 mice per group for B16F10-Tb model and n = 3 per group for K7M2-Tb model. n,o, Representative images of BODIPY staining of the livers from B16F10- and K7M2-Tb mice (n), and B16F10-CL-EVP- and K7M2-CL-EVP-educated mice (o), treated with anti-TNF antibody or IgG1 isotype control (see also Fig. 4d, e). Scale bars, 20 µm. P values were determined by the one-way ANOVA with post hoc Tukey’s test (b), or two-tailed, unpaired Student’s t-test (g-i,m). Data are mean ± s.e.m. NS, not significant. For cytokine array blot source data of (c-f), see Supplementary Fig. 2.

Source Data