Fig 2: Stem-like properties of CCSCs were inhibited by miR-195-5p/497–5p through negative regulation of MCM2. a, Quantitation for the formation ability of tumor microspheres of CCSCs as detected by tumor sphere self-renewal assay. b, Quantitation for colony formation ability of cells as tested by soft agar colony formation assay. c, Protein expression of stem-like properties-related proteins (CD133 and EpCAM) as detected by Western blot analysis. d, Protein expression of stem cell markers (Nanog, Oct-4 and Sox-2) as detected by Western blot analysis. Measurement data were expressed as mean ± s.d. Data among multiple groups were compared using one-way analysis of variance and then analyzed with Tukey’s post-hoc test. Cell experiment was repeated three times. * p < 0.05 vs. si-NC. # p < 0.05 vs. pcDNA-MCM2 + miR-NC

Fig 3: PF Contains Nanoparticles of the Size and Antigenic Profile of Exosomes(A and B) Nanoparticle tracking analysis (NTA) was used to quantify the concentration (A) and size distribution (B) of particles in the total PF and plasma. The dotted rectangles evidence exosome-sized vesicles (30–120 nm). (C and D) PF and plasma exosome preparations were positive for the exosomal markers ALIX, FLOT1, EpCAM and CD63. Transmission electron microscopy (TEM) using a gold particle-conjugated anti-CD63 antibody further confirmed exosome identity. In the bottom right corner of (D), next to CD63-immunoreactive exosomes, is shown a larger unreactive cytoplasmic vesicle as negative control (the scale bar represents 100 nm). Unpaired two-tailed Student’s t test was applied. All values are mean + SEM; n = 5.

Fig 4: NF-?B activation downregulates miR-195-5p/497–5p and thereby promotes tumorigenesis and stem-like properties of CCSCs in vivo. a & b, CCSCs were treated with overexpressed miR-195-5p/497–5p and then subcutaneously injected into nude mice to establish a subcutaneous xenograft tumor model. At the same time, mice were injected with LPS or PBS twice a week until the day before the end of the experiment. The volume and weight of xenografted tumors were observed and recorded (n = 6 for each group). c, RNA was extracted from tumor tissues and the expression of miR-195-5p and miR-497-5p was detected by RT-qPCR. d, The protein expression of p65, extent of p65 phosphorylation, CD133, Nanog, Oct-4, Sox-2, and EpCAM in tumor tissues as detected by Western blot analysis. e, The protein expression of apoptosis-related proteins (Bcl-2 and Bax) in tumor tissues as detected by Western blot analysis. (1, miR-NC + PBS; 2, miR-NC + LPS; 3, miR-497-5p/195-5p agomir + PBS; 4, miR-497-5p/195-5p agomir + LPS). Measurement data were expressed as mean ± s.d. Data between two groups were compared using unpaired t-test. Data among multiple groups were compared using one-way analysis of variance and then analyzed with Tukey’s post-hoc test. Repeated measures analysis of variance was used for comparing data among multiple groups at different time points, followed by Bonferroni post-hoc test. Cell experiment was repeated three times. * p < 0.05 vs. miR-NC + PBS. # p < 0.05 vs. miR-497-5p/195-5p agomir + PBS

Fig 5: Periostin promotes exosome secretion from pancreatic cancer cells that induce EMT.A Electron microscopy images of exosomes isolated from SW1990 and CFPAC-1 cells treated with or without human recombinant Periostin protein (PN). B Concentration and size analysis of SW1990-exo and CFPAC-1-exo isolated by ultracentrifugation with or without PN. C, D Concentration and diameter analysis of SW1990-exo and CFPAC-1-exo treated with or without PN. The experiment was repeated three times, and the significance was analyzed using a Student’s t test. Data are shown as mean ± SD (*P < 0.05 and ** P < 0.01 vs. control). E Western blot analysis for exosomal proteins Alix, Annexin-V, CD54, CD9, GM130, EpCAM, and Flotillin-1 treated with or without PN in SW1990 and CFPAC-1 cells. β-actin was used as a loading control. F Expression of exosomal CD54 was examined by IHC in PDAC tissues. G The EMT effect was further confirmed by western blot analysis of epithelial or mesenchymal markers in SW1990 and CFPAC-1 cells treated with or without PN. H The EMT effect was validated by immunofluorescence analysis in SW1990 cells treated with or without PN.