Fig 2: Long-term adaptation to PA phenocopy obesity-induced stem-cell features.a Distributions of relative barcode frequency for three tumors in each group. b Apoptotic rate of parental and adapted HCC1806 cells which were treated with 400 µM PA and vehicle (Ctrl) for 48 h. Both early (Annexin V+/PI-) and late (Annexin V+/PI+) apoptotic cells were included for the apoptotic rate % calculation. Data are represented as mean ± SEM of 3 replicates. c Time-dependent proliferation assay of parental and adapted HCC1806 following 48 h. Cells were exposed to 400 µM PA and vehicle (Ctrl). Cell growth was determined by high content imaging and represented as % confluence normalized to t = 0. For each time point, data are represented as mean ± SEM of 6 replicates. d Representative contour plots of mass cytometry data colored by density of cells showing the changes between parental and adapted HCC1806 cells. Color code represents the cell density from low (blue) to high (red). e Representative tSNE plots of single parental and adapted HCC1806 cells colored by expression of CD133, Axl, and CD44. f Tumor incidence following orthotopic implantation of the indicated number of parental and adapted E0771 cells into chow and HFD-fed mice. The frequency of cancer stem-like cells was calculated by the extreme limiting dilution analysis. The default Chisquare test in ELDA was performed to evaluate the differences between parental and adapted cells (NS, P value >0.05). g The distribution of genes induced by obesity (obese and overweight compared to non-obese patients) in PM hormone negative breast cancers patients among the gene expression changes observed in PA-adapted cell lines. We have included the mutual information value (MI) and its associated z-score reported by iPAGE. For visualization, the enrichment/depletion of the query gene-set was determined using the hyper-geometric test and the resulting p-value was used to define an enrichment score that is shown as a heatmap across the expression bins. The obesity-induced genes were significantly enriched in the top-most bin. The red and blue borders in the heatmap denoted statistical significance for enrichment and depletion respectively. Source data are provided as a Source data file.

Fig 3: Obesity is associated with increased frequency of stem cell-like cancer cells in PM/ER-/PR- breast cancer patients and mouse models of breast cancer.a Tumor incidence following orthotopic implantation of cancer cells into the mammary fat pads of chow- and HFD-fed mice. b Kaplan–Meier curves show disease-specific survival for PM/ER-/PR- patients (n = 48) with high (red) or low (blue) BMI. Log-rank (Mantel–Cox) P value is denoted for difference in disease-specific survival. c, d Representative tissue microarray and QuPath analysis mask pictures of CD133 (c) and Axl (d) staining in high (BMI > 25, n = 23 for CD133; n = 21 for Axl) or low (BMI = 25, n = 13 for CD133; n = 11 for Axl) BMI PM/ER-/PR- patients’ tumor samples, stroma is marked in green, CD133- or Axl-/low cancer cells are marked in blue and positive staining cancer cells are marked in red (for c, P = 0.0245; d, P = 0.023). e Time-dependent proliferation assay of ex vivo E0771 cells isolated from chow or HFD-fed mice. For each time point, data are represented as mean ± SEM of four tumor samples (eight replicates per tumor sample were measured) from each group. f Tumorsphere formation assay of E0771 ex vivo cells isolated from chow diet or HFD-fed mice. Representative images of day-5 tumorspheres formation of E0771 ex vivo cells and red arrowheads mark the identified tumorspheres. Quantification of day-5 tumorspheres is represented as mean ± SEM of four tumor samples from each group and three replicates were measured for each sample, P = 0.0002. g Fatty acid and glucose oxidation on E0771 ex vivo cells isolated from two chow diet-fed mice and three HFD-fed mice. The oxidation data are normalized to cell protein content. For each sample, 8 replicates are measured and data are represented as mean ± SEM of all replicates from each group. Fatty acid oxidation P = 0.0002, Glucose oxidation P = 0.0365. For c, d, unpaired, two-tailed Welch’s t-test was used for statistical testing. For f, g, statistical significance determined with unpaired, two-tailed Student’s t-test (*P value <0.05; ***P value <0.001). Source data are provided as a Source data file.

Fig 4: Circulating agrin-positive EVs are a biomarker of disease progression and predict response to treatment in patients with PDAC. (A) Presurgery and postsurgery analysis of the percentage of circulating agrin-positive EVs coupled to beads in the serum of PDAC patients (n=19, paired t-test; *p<0.05). (B, C) Prechemotherapy and postchemotherapy analyses of the percentage of circulating agrin-positive EVs coupled to beads in the serum of patients with PDAC treated with all regimens (B) (n=24, paired t-test; *p<0.05) and folfirinox-based regimens (C) (n=13, paired t-test; **p<0.01). (D) Correlation between the percentage of circulating CD133-positive EVs coupled to beads and the log10 of the percentage of agrin-positive EVs coupled to beads in the serum of patients with PDAC (n=106, Spearman r=0.6272). (E) Analysis of the percentage of circulating agrin-positive EVs coupled to beads in the serum of three patients with PDAC throughout time. QRT, chemotherapy, TAborted surgery due to non-resectable tumour. (F) Receiver operating curve analysis for the percentage of agrin-positive (red), CD133-positive (blue), and combination of agrin-positive and CD133-positive (purple) EVs coupled to beads and CA19-9 (green) in the serum of patients with PDAC not submitted to surgery (n=22 in circulating agrin-positive and CD133-positive EVs coupled to beads analysis and n=20 in CA19-9 analysis). (G) Receiver operating curve analysis for the percentage of agrin-positive (red), CD133-positive (blue), and combination of agrin-positive and CD133-positive (purple) EVs coupled to beads and CA19-9 (green) in the serum of patients with PDAC not submitted to surgery and treated with chemotherapy (n=17). In tables: a means under the non-parametric assumption and b means null hypothesis: true area=0.5. EVs, extracellular vesicles; PDAC, pancreatic ductal adenocarcinoma.

Fig 5: Agrin is enriched in CSC EVs. (A) Experimental approach to perform LC/ESI–MS/MS in subpopulations of cells and respective EVs (CD24+CD44+, CD133+, CD24-CD44+, 4N and EpCAM+) in four human PDAC cell lines (BxPC3, PANC-1, T3M4 and MIA PaCa-2) for a total of 38 samples (MIA PaCa-2 cells are EpCAM-). (B) Heatmap depicting protein clusters present in PDAC subpopulations and respective EVs in four cell lines. Dendogram displays unsupervised hierarchical clustering showing separation of three protein clusters: EVs, cells and cells+EVs. The full heatmap is shown in online supplemental figure 7D. (C) Functional enrichment analysis of protein clusters. Dot plot representing the top 6 reactome-enriched pathways per cluster (adjusted p value <0.05).Gene ratio corresponds to the relative size of every pathway in each protein cluster. (D) Venn diagram of total proteins detected in CSC and NSCC EVs isolated from four PDAC cell lines. Edges represent the number of proteins detected only in CSC or NSCC EVs, and intersection represents the number of proteins common to both subpopulations. (E) Top 5 enriched reactome pathways in CSC, NSCC and in both subpopulations of EVs (adjusted p value <0.05). (F) Gene set enrichment analysis demonstrates that proteins in the axon guidance pathway separate CSC EVs from NSCC EVs. (G) Venn diagram depicting the intersection of the proteins found in CSC EVs across the four PDAC cell lines. Out of the 233 proteins common across all CSC EVs, 79 correspond to proteins of the axon guidance pathway. Out of these, 14 were present in CSC EVs across all four cell lines. These 14 were ranked by average DESeq2 normalised peptide counts (table on the right). (H) FACS analysis and representative histogram plots of agrin-positive EVs derived from MIA PaCa-2 NSCC and CSC subpopulations (n=4, Mann-Whitney test, *p<0.05). Data are mean±SEM. CSC, cancer stem cells; EVs, extracellular vesicles; GTP, Guanosine-5'-triphosphate; HSC, hematopoietic stem cells; IGFBP, Insulin-like growth factor binding protein; LC/ESI–MS/MS, liquid chromatography–electrospray ionisation–tandem mass spectrometry; NSCC, non-stem cancer cell; PDAC, pancreatic ductal adenocarcinoma; PKN, protein kinase N; RHO, ras homologous; ROBO, roundabout guidance receptor; SLIT, slit guidance ligand; SRP, signal recognition particle.