Fig 2: 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.

Fig 3: The most frequent communication route in the PDAC EVNet occurs from CSC to NSCC. (A) Schematics of the methodological approach: MIA PaCa-2 clones expressing different fluorescently labelled markers are cultured at the percentages found in the parental cell line and are analysed through flow cytometry in order to trace the flow of EVs among subpopulations. A total of seven intermix of colour-coded subpopulations was analysed, corresponding to five distinct intermixes. Subpopulations: CD24+CD44+ refers to CD24+CD44+CD133-EpCAM-; CD133 +refers to CD133+CD24-CD44-EpCAM-; CD24-CD44+ refers to CD24-CD44+CD133-EpCAM-; 4N refers to CD24-CD44-CD133-EpCAM-. (B) Representative confocal microscopy live images of 72-hour culture MIA PaCa-2 colour-coded subpopulations (CD133+ CD63-GFP, CD24+44+ Rab5-mPlum, CD24-CD44+ CD82-eYFP and 4N CD81-tdTomato). Right panel: representative images of a cell where EVs uptake was not detected (single positive), a cell that received EVs from one different subpopulation (double positive), a cell that received from two other subpopulations (triple positive) or from all four subpopulations (quadruple positive). Scale bars from left to right: 20, 10 and 10 µm. (C) Dot plot representing the percentage of single-positive, double-positive, triple-positive and quadruple-positive cells found in the cocultures (n=7, Mann-Whitney test ***p<0.001). (D) Quantification of the percentage of cells that received EVs from CD133+, CD24+CD44+, CD24-44+ and 4N subpopulations (n=7, one-way analysis of variance (ANOVA) *p<0.05, **p<0.01, ****p<0.0001). (E) Quantification of cells of different subpopulations that receive EVs from CSC or NSCC (highlighted on top). The fold change was calculated against the minimum percentage of communication found in each coculture (n=7). (F) Quantification of the percentage of cells that received EVs (left) and schematic representation of the most frequent communication route in the EVNet, from CSC to NSCC (right, comparison of CSC positive for NSCC EVs and NSCC positive for CSC EVs; n=7, Mann-Whitney test, *p=0.0379). Data are mean±SEM. CSC, cancer stem cell; EVs, extracellular vesicles; EVNet, Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network; NSCC, non-stem cancer cells; PDAC, pancreatic ductal adenocarcinoma.

Fig 4: Stem cell mobilization with MRG-001Subjects received SC injections every other day of saline (placebo control), mid-dose (0.01 mL/kg), or high-dose (0.02 mL/kg) MRG-001 (n = 4/group). Venous blood was collected and PBMCs were isolated at different time intervals on day 1 after a single dose SC injection of saline or MRG-001 and on day 5 after the third dose. Stem cell populations (CD45IntCD34+, CD45IntCD34+CD90+, CD45IntCD34+CD133+, CD45IntCD34+CD133+CD31+, CD45IntCD34+CD133+VEGFR2+, CD45IntCD34+SSEA3+) in PBMC were analyzed by flow cytometry. At each blood draw timepoint, absolute number of circulating stem cells, such as CD45IntCD34+ HSCs, was calculated by multiplication of CD45IntCD34+ cell fraction of PBMC by the total circulating PBMC count.(A) Stem cells in peripheral blood on day 1 after a single dose SC injection of saline or MRG-001.(B) Stem cells in peripheral blood on day 5 after the third dose. Each value represents the mean ± SD.Yellow marked cells represent significant difference with p < 0.05.