Extended Data Fig. 5: Multi-omics integration leading to interleukin-6.

(a-j) Transcriptomic (RNA-seq) analysis of metabolic tissues (liver, eWAT, iWAT, heart, GC muscle) from healthy controls (PBS-injected, no tumour, Ctrl), non-cachectic (NC26 tumours, Non-cax), pre-cachectic (C26 tumours, Pre-cax) and cachectic (C26 tumours, Cax) mice (n = 4 animals per group, same animals as in Figs. 1–3). See also Fig. 4. Significant genes are defined by an adjusted p value < 0.05 and log2 fold change > 0 or < 0. (a-e) Venn diagrams showing the number of genes significantly altered in Non-cax, Pre-cax and Cax mice compared to Ctrl in liver (a), eWAT (b), iWAT (c), heart (d) and GC muscle (e). (f) Volcano plots showing the number of genes positively and negatively altered in Cax mice vs. Ctrl. Data presented as log2 fold change (Cax/Ctrl) and adjusted p values. Significant genes with an adjusted p value < 0.05 are highlighted in colored fields on each plot. (g) Venn diagram showing the number of genes significantly altered in Cax mice vs. Ctrl in the different metabolic tissues studied. (h) Top pathways altered in a similar manner in cachexia target tissues (liver, eWAT, iWAT, heart, GC muscle) in Cax vs. Non-cax mice. Data are represented as top z-scores: pathways predicted to be activated in red and inhibited in blue (IPA, Qiagen). (i) Top pathways commonly altered in both transcriptomics and metabolomics datasets based on p value (IPA, Qiagen) in Cax vs. Non-cax mice. (j) Top potential upstream regulators of observed changes in transcriptomics and metabolomics common to the different metabolic tissues of Cax mice vs. Non-cax mice (IPA, Qiagen). Data are represented as top significant pathways based on p value. (k-r) Mice were injected either with PBS (healthy controls, Ctrl, grey), control C26 cancer cells (C26-scramble, dark red), or C26 cancer cells with an IL6 knock-out (C26-IL6^KO, orange). n = 3 animals per group. (k) Secretion of IL6 from control C26-scr and C26-IL6^KO tumour cells (n = 1 replicate), (l) IL6 concentrations in C26-scr and C26-IL6^KO tumour lysates (n = 3 animals per group), and (m) IL6 levels in plasma of C26-scr and C26-IL6^KO tumour bearing mice (n = 3 animals per group). (n-o) Kinetic of tumour growth (n) and final tumour weights (o). (p) Body weight loss, expressed as percentage of initial body weight. (q) Final tissue weights. (r) Relative mRNA expression level of key enzymes of one-carbon metabolism and related pathways in eWAT. Data expressed as fold change of Ctrl. (s-u) Mice were injected with PBS or C26 cancer cells and treated with a control IgG antibody or a neutralising IL6 antibody. From left to right: healthy controls (PBS-injected, Ctrl, grey), cachectic C26 tumour mice (C26, dark red), cachectic C26 tumour mice treated with control IgG (C26 + IgG, light red), C26 tumour mice treated with an IL6 neutralising antibody (C26 + IL6-nAB, orange). n = 8 animals per group. Relative mRNA expression of key enzymes of one-carbon metabolism and related pathways in liver (s), GC muscle (t) and eWAT (u). Data expressed as fold change of Ctrl. Statistical analysis on raw data (2^−ΔCt values) (r-u). Data are mean ± s.e.m. Statistical analysis: unpaired, non-adjusted, Student’s t test (l, o), unpaired one-way ANOVA with Tukey’s post-hoc tests (p-r), unpaired one-way ANOVA with Dunnett’s or Kruskal Wallis with Dunn’s post-hoc tests (s-t, vs. C26). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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