Journal: Nature Metabolism

Article Title: Multi-omics profiling of cachexia-targeted tissues reveals a spatio-temporally coordinated response to cancer

doi: 10.1038/s42255-025-01434-3

Figure Lengend Snippet: a , A schematic overview of the experimental workflow. Mice were divided into four groups: mice injected with PBS, healthy controls, no tumour (Ctrl, grey, no weight loss); mice injected with NC26 cancer cells, non-cachectic tumour controls (Non-cax, blue, no weight loss); mice injected with C26 cancer cells and killed before onset of weight loss, pre-cachectic tumour mice (Pre-cax, light red, no weight loss); mice injected with C26 cancer cells and killed once they developed cachexia, cachectic tumour mice (Cax, dark red, mean body weight (BW) loss of 10%). On the day of euthanasia, mice were fasted for 6 h and injected with an isotopic tracer ([ 13 C 6 ]-glucose). Tissues (plasma, liver, eWAT, iWAT, heart, GC muscles, soleus and tumour) were collected exactly 1 h later. Tissues were then processed for tracer metabolomics and results submitted to bioinformatics. n = 4 animals per group. See also Extended Data Figs. and . b , Kinetics of body weight loss expressed as a percentage of initial body weight. c , Final tumour weight. Data are mean ± s.e.m. Statistical analysis: paired two-way ANOVA with Dunnett’s post-hoc tests versus Ctrl ( b ) and unpaired Kruskal–Wallis with Dunn’s post-hoc test ( c ). d , Total number of metabolites per tissue included in the analysis after filtering . See also Supplementary Table (sum of all isotopologues; log-transformed imputed, scaled data) and our WebApp ( https://m3cav.metabolomics.fgu.cas.cz/ ). e – l , PLSDA score plots of samples based on metabolites log-transformed imputed and scaled data for each organ, tumour and plasma; see icon legend in a . Ellipses represent 95% confidence intervals. m , Number of metabolites significantly altered in the time course of cachexia development. Grey: unchanged in Non-cax, Pre-cax and Cax versus Ctrl. Blue: significant in Non-cax versus Ctrl. Light red: significant in Pre-cax versus Ctrl. Dark red: significant in Cax versus Ctrl. List of significantly different metabolites per tissue can be found in Supplementary Table . n , Heatmaps based on hierarchical clustering of all metabolites (Extended Data Fig. ), which are significantly altered in at least one metabolic tissue of Cax mice, manually organised per metabolite class. Data are represented as log fold change (FC) (tumour group/controls). Tissues from left to right: plasma, liver, eWAT, iWAT, heart, GC muscle, soleus muscle, tumour. Groups from left to right: blue, Non-cax/Ctrl; light red, Pre-cax/Ctrl; dark red, Cax/Ctrl. Tumour: light red Pre-cax/Non-cax, dark red Cax/Non-cax. A list of metabolites and associated classes can be found in Supplementary Table . m , n , Statistical analysis of filtered data: one-way ANOVA following post-hoc correction based on Tukey’s honestly significant difference procedure. Panel a and icons in e – i , k and l created with BioRender.com ; icon in j reproduced from Servier Medical Art ( https://smart.servier.com/ ) under a Creative Commons license CC BY 4.0.

Article Snippet: Eight mice were randomly assigned to four groups based on their body weight on the day of cell injection: Ctrl (sham-injected), C26 (C26 cancer cells and treated with PBS), C26 + IL6-nAB group (C26 cancer cells and treated with 300 μg monoclonal rat anti-murine IL6 antibody (clone MP5-20F3, BioXCell)) and C26 + IgG group (C26 cancer cells and treated with 300 μg rat IgG1 isotype control (cat. no. BE0088, BioXCell)).

Techniques: Injection, Clinical Proteomics, Muscles, Transformation Assay

Journal: Nature Metabolism

Article Title: Multi-omics profiling of cachexia-targeted tissues reveals a spatio-temporally coordinated response to cancer

doi: 10.1038/s42255-025-01434-3

Figure Lengend Snippet: ( 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. – ). See also Fig. . 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.

Article Snippet: Eight mice were randomly assigned to four groups based on their body weight on the day of cell injection: Ctrl (sham-injected), C26 (C26 cancer cells and treated with PBS), C26 + IL6-nAB group (C26 cancer cells and treated with 300 μg monoclonal rat anti-murine IL6 antibody (clone MP5-20F3, BioXCell)) and C26 + IgG group (C26 cancer cells and treated with 300 μg rat IgG1 isotype control (cat. no. BE0088, BioXCell)).

Techniques: RNA Sequencing, Injection, Control, Knock-Out, Clinical Proteomics, Expressing

Journal: Nature Metabolism

Article Title: Multi-omics profiling of cachexia-targeted tissues reveals a spatio-temporally coordinated response to cancer

doi: 10.1038/s42255-025-01434-3

Figure Lengend Snippet: Transcriptomic analysis of cachexia target tissues from Ctrl, Non-cax and Cax tumour mice. See also Fig. for the experimental set-up, and Extended Data Fig. . n = 4 animals per group. a , Top pathways altered in a similar manner in cachexia target tissues (liver, eWAT, iWAT, heart and GC muscle) from Cax versus Ctrl mice. Data are represented as top z scores: pathways predicted to be activated in red and inhibited in blue (IPA, Qiagen). b , Top pathways commonly altered in both transcriptomics and metabolomics datasets based on P value (IPA, Qiagen) in Cax versus Ctrl mice. Full pathway lists can be found in Supplementary Fig. . See also Extended Data Fig. for similar analyses in Cax versus Non-cax. c – f , Heatmaps showing the changes in mRNA expression of enzymes involved in one-carbon metabolism and related metabolic pathways (methionine cycle ( c ), methyltransferases ( d ), glutathione metabolism ( e ) and urea cycle ( f )). Data from RNA sequencing analysis, presented as log 2 fold change (Cax/Ctrl and Cax/Non-cax) and adjusted P values. * P < 0.05. Ahcy , adenosylhomocysteinase; Amd , S -adenosylmethionine decarboxylase; Arg , arginase; Asl , arginosuccinate lyase; Ass , arginosuccinate synthetase; Bhmt , betaine-homocysteine S -methyltransferase; Cbs , cystathionine beta-synthase; Cth , cystathionine gamma-lyase; Dnmt , DNA (cytosine-5)-methyltransferase; Gclc , glutamate-cysteine ligase catalytic subunit; Gnmt , glycine N -methyltransferase; Gpx , glutathione peroxidase; Gss , glutathione synthetase; Gst , glutathione S -transferase; Kmt , lysine (K)-specific methyltransferase; Mat , methionine adenosyltransferase; Mgst , microsomal glutathione S -transferase; Mtap , methylthioadenosine phosphorylase; Mthfr , methylenetetrahydrofolate reductase; Mtr , 5-methyltetrahydrofolate-homocysteine methyltransferase; Mtrr , 5-methyltetrahydrofolate-homocysteine methyltransferase reductase; Nnmt , NAM N -methyltransferase; Odc , ornithine decarboxylase; Otc , ornithine transcarbamylase; Paox , polyamine oxidase; Pemt , phosphatidylethanolamine N -methyltransferase; Prmt , protein arginine N -methyltransferase; Sat , spermidine/spermine N1-acetyltransferase; Shmt , serine hydroxymethyltransferase; Sms , spermine synthase; Srm , spermidine synthase. See also Supplementary Fig. for visual integrations of transcriptomics and metabolomics data in Cax tissues. g , Top potential upstream regulators of observed changes in transcriptomics and metabolomics common to the different metabolic tissues of Cax versus Ctrl mice (IPA, Qiagen). Data are represented as top significant pathways based on P value. h – k , Relative mRNA expression levels of key enzymes ( h and i ) and metabolites ( j and k ) of one-carbon metabolism and related pathways in liver ( h and j ) and GC muscle ( i and k ) from healthy controls (PBS-injected, grey), C26-control tumour mice (C26-scramble (scr), dark red) and C26-IL6-knock out tumour mice (C26-IL6 KO, orange). Metabolite IDs as in the list presented in Fig. . n = 3 animals per group. Data are the mean ± s.e.m. In h – k , statistical analysis on raw data (2 −ΔCt values and MS signal intensities, arbitrary units (AU)) was performed using one-way ANOVA with Tukey’s post-hoc tests or Kruskal–Wallis with Dunn’s post-hoc tests. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Eight mice were randomly assigned to four groups based on their body weight on the day of cell injection: Ctrl (sham-injected), C26 (C26 cancer cells and treated with PBS), C26 + IL6-nAB group (C26 cancer cells and treated with 300 μg monoclonal rat anti-murine IL6 antibody (clone MP5-20F3, BioXCell)) and C26 + IgG group (C26 cancer cells and treated with 300 μg rat IgG1 isotype control (cat. no. BE0088, BioXCell)).

Techniques: Expressing, RNA Sequencing, Injection, Control, Knock-Out

Journal: Nature Metabolism

Article Title: Multi-omics profiling of cachexia-targeted tissues reveals a spatio-temporally coordinated response to cancer

doi: 10.1038/s42255-025-01434-3

Figure Lengend Snippet: ( a-e ) C2C12 myotubes were treated with different doses of L-methionine (0 µM, 20 µM, 100 µM) for 48 h. See also Fig. . ( a-e ) Incorporation of labelled carbons from [ 13 C 6 ]-glucose into metabolites of the TCA cycle ( n = 3 replicates per group). Unlabelled metabolites are referred as M + 0, isotopically-labelled metabolites as M + X. Data are presented as MS signal intensities (arbitrary units A.U.). ( f-j ) C2C12 myotubes were treated with different doses of FIDAS-5 (methionine adenosyltransferase inhibitor) for 48 h. ( f ) Relative levels of substrates and products of one-carbon metabolism, presented as fold change of vehicle condition ( n = 8 replicates per group). ( g-h ) Representative images ( g ) and quantification of myotube diameters ( h ) ( n = 8 replicates per group). ( i-j ) Glucose levels ( n = 14 replicates per group) ( i ) and pH of culture media ( j ) ( n = 9 replicates per group). ( k-q ) C2C12 myotubes were treated with 100 ng/mL of recombinant IL6 and different doses of FIDAS-5 for 48 h. ( k-l ) Validation of the activation of rIL6 signalling cascade via pY705-STAT3 protein levels ( n = 3 replicates per group, representative experiment out of 3 independent experiments). ( m ) Relative levels of substrates and products of one-carbon metabolism, presented as fold change of vehicle condition ( n = 6 replicates per group). ( n-o ) Representative images ( n ) and quantification of myotube diameters ( o ) ( n = 7 replicates per group). ( p-q ) Glucose levels ( i ) and pH of culture media ( j ) ( n = 7 replicates per group). ( r-t ) 3T3-L1 adipocytes were treated with different doses of L-methionine (0 µM, 20 µM, 100 µM) for 24-48 h. ( r-s ) Glycerol ( n = 10 replicates per group) ( s ) and NEFA ( t ) release after 24 h incubation with L-methionine (readouts for lipolytic activity). NEFA release was measured in the presence or absence of isoproterenol to assess stimulated and basal lipolysis, respectively ( n = 5 replicates per group). ( t ) Glucose levels of culture media after 48 h of incubation (n = 10 replicates per group). Data are mean ± s.e.m. Statistical analysis: unpaired two-way ANOVA with Dunnett’s post-hoc tests ( a - e , vs . 0 µM), unpaired one-way ANOVA with Dunnett’s post-hoc tests or Kruskal-Wallis with Dunn’s post-hoc tests ( f - j , vs . 0 µM; l, vs . Ctrl vehicle; m - q , vs . rIL6 vehicle). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs . 0 µM or vehicle ( a - j , r - t ), Ctrl + vehicle ( l ) or vs . rIL6 + vehicle ( m - q ).

Article Snippet: Eight mice were randomly assigned to four groups based on their body weight on the day of cell injection: Ctrl (sham-injected), C26 (C26 cancer cells and treated with PBS), C26 + IL6-nAB group (C26 cancer cells and treated with 300 μg monoclonal rat anti-murine IL6 antibody (clone MP5-20F3, BioXCell)) and C26 + IgG group (C26 cancer cells and treated with 300 μg rat IgG1 isotype control (cat. no. BE0088, BioXCell)).

Techniques: Recombinant, Biomarker Discovery, Activation Assay, Incubation, Activity Assay