Extended Data Fig. 5: Tumour cell line (CL)-derived EVPs dysregulate liver metabolism.

a, Representative TEM images of B16F10-CL-EVPs (left) and K7M2-CL-EVPs (right). Scale bar, 200 nm. This experiment was repeated three times independently with similar results. b, NTA of B16F10-CL-EVPs and K7M2-CL-EVPs. Shown are graphs representative of three independent experiments. c,d, Representative images (left) and associated statistical analysis of relative signal intensity (right) of the livers from mice 24 h post intravenous injection of 10 µg of CellVue NIR815-labeled B16F10-CL-EVPs (c) and K7M2-CL-EVPs (d), and their mock PBS-injected controls. n = 6 mice injected with B16F10-CL-EVPs or PBS mock reaction; n = 7 mice injected with K7M2-CL-EVPs and n = 6 mice injected with PBS mock control. e, Schematic illustration of syngeneic mouse education with B16F10-CL-EVPs or K7M2-CL-EVPs, and PBS control. f, PCA of gene expression in the livers from mice educated with B16F10-CL-EVPs (left) and K7M2-CL-EVPs (right), compared to PBS-educated controls. n = 5 mice per group for B16F10-CL-EVP education model; n = 3 mice per group for K7M2-CL-EVP education model. g,h, GSEA of gene expression profiles, ranked based on the sign of log₂FC∗(-log₁₀P value), in the livers from B16F10-CL-EVP- (g) and K7M2-CL-EVP- (h) educated mice, compared to PBS-educated controls, using hallmark gene sets. Significantly changed signaling pathways with nominal P < 0.05 are shown. n = 5 B16F10-CL-EVP-educated mice and controls; n = 3 K7M2-CL-EVP-educated mice and controls. Gene lists for signaling pathways are shown in Supplementary Tables 22 and 23. i, Heatmap showing the metabolites significantly changed in the livers of B16F10-CL-EVP-educated mice, compared to PBS-educated controls. n = 5 control and n = 7 B16F10-CL-EVP-educated mice. Metabolomic mass spectrometry data are shown in Supplementary Table 29. j,k, Volcano plots showing the significantly (P < 0.05) enriched lipid classes (red) (j) and the significantly (P < 0.05) enriched triglyceride species (red) (k) in the livers of B16F10-CL-EVP-educated mice, compared to PBS-educated controls. n = 5 control and n = 6 B16F10-CL-EVPs educated mice. Lipidomic mass spectrometry data are shown in Supplementary Table 30. l, Heatmap showing the metabolites significantly changed in the livers of K7M2-CL-EVP-educated mice, compared to PBS-educated controls. n = 5 K7M2-CL-EVP-educated mice and controls. Metabolomics MS data are shown in Supplementary Table 31. m,n, Volcano plots showing the significantly (P < 0.05) changed lipid class (blue) (m), and the significantly (P < 0.05) enriched triglyceride species (red) (n) in the livers from K7M2-CL-EVP-educated mice, compared to PBS-educated controls. n = 5 control and n = 4 K7M2-CL-EVP-educated mice. Lipidomic mass spectrometry data are shown in Supplementary Table 32. o,p, Representative immunofluorescence (IF) images (left) and associated statistical analysis (right) of BODIPY staining of the livers from B16F10-CL-EVP- (o) and K7M2-CL-EVP- (p) educated mice, and PBS-educated controls. n = 4 mice per group for B16F10-CL-EVP-education model; n = 5 mice per group for K7M2-CL-EVP-education model. Scale bars, 20 µm. q, Quantification of BODIPY staining of the livers from B16F10- and K7M2-conditioned media -educated mice, compared to blank DMEM media-educated mice. n = 5 mice per group for B16F10-conditioned media model and n = 4 mice per group for K7M2-conditioned media model. r, Quantification of BODIPY staining of the livers from PBS-educated and B16F10-CL-EVP-educated mice, in the absence or presence of B16F10-conditioned media co-education. n = 4 mice educated with PBS, n = 5 mice educated with B16F10-CL-EVPs alone, and n = 4 mice educated with B16F10-CL-EVPs together with B16F10-conditioned media. P values were determined by the two-tailed, unpaired Student’s t-test for (c,d,j,k,m-p) and one-way ANOVA with post hoc Tukey’s test for (r). Data are mean ± s.e.m. NS, not significant. CM, conditioned media.

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