Fig 2: Neutrophil dysfunction and organ damage due to sepsis. (A) Agarose chemotaxis model to measure neutrophil chemotaxis distance, chemotaxis index. (B) Chemotaxis distance of sepsis patients was significantly smaller than that of normal controls (p<0.05). (C) PMA stimulation of neutrophils in sepsis patients and normal subjects, respectively, and flow cytometry detection of intracellular ROS formation. The ability of neutrophils to produce ROS was significantly lower in septic patients than in normal subjects (p<0.05). (D) Neutrophils from normal and septic patients were extracted separately and cultured in vitro for 24 h, and the apoptosis rate was detected by flow cytometry. Apoptosis of neutrophils was significantly delayed in sepsis patients compared with normal subjects. (E) Plasma from septic patients and normal subjects were isolated and stimulated separately with normal human neutrophils, and sytox neutrophil DNA staining (scale bar: 100 µm). Sepsis model of CLP mice (n=5) was established. (F) PMA (100 nM) stimulated neutrophils for 30 min, and ROS production was detected by flow cytometry. (G) PMA (100 M) stimulated neutrophils for 30 min, and cell membrane CD63 expression was detected by flow cytometry. (H) Ly6g-positive cells in lung sections from both groups were detected by immunohistochemistry (scale bar: 50 µm). (I) The infiltration of neutrophils (ly6g +) in the lungs of septic mice was increased compared with the normal mice (p < 0.05). (J) In alveolar lavage fluid, CLP mice had increased neutrophils compared with normal mice (p < 0.05). K: Immunofluorescence was performed for citrullinated histones and MPO citrullinated histones

Fig 3: Cancer cell-derived sEVs can stimulate endothelial cell migration and tube formation independently of uptake. a Immunoblot of TSG101 and flotillin-1 in fractions of the indicated buoyant densities that were isolated from media conditioned by ovarian (ES2), colorectal (HCT116), and renal (786-0) cancer cell lines, and from ovarian cancer patient ascites. b Immunogold labeling of CD63 on vesicles in sEV fractions (i.e., density of 1.09–1.13 g/mL). Scale bar = 100 nm. c–f Human umbilical vein endothelial cells (HUVEC) were pretreated with endocytosis inhibitors (chlorpromazine, CPZ; dynasore, DYN) or with dimethyl sulfoxide (DMSO) solvent, and then stimulated with sEVs of ES2, HCT116, and 786-0 cells. Shown are numbers (c) and representative images (d) of migrating HUVEC at 5 h after stimulation, and numbers (e) and representative images (f) of tubes formed at 4 h after stimulation. Mean ± SD of n = 4 independent experiments are shown. Scale bar = 100 µm. g, h HUVEC were treated as in c and evaluated for uptake of PKH26 dye-labeled sEVs by flow cytometry at 5 h thereafter. Shown are mean fluorescence intensity (MFI) values of PKH26 fluorescence detected in HUVEC in n = 3 independent experiments (g) and representative histogram plots (h). Gating strategy and contour plots are shown in Supplementary Fig. 3b and 4b, respectively. **P < 0.01, ***P < 0.001, ****P < 0.0001, by ANOVA with Bonferroni’s corrections; one-way in g, two-way in c and e. ns: not significant. Source data used for graphs in c, e, and g can be found in Supplementary Data 1

Fig 4: Characterization and function of exosomes derived from G-MDSCs. (A) Transmission electron microscopy images of lung cancer G-MDSC-derived exosomes; (B) NanoSight measurement of particle size distribution and concentration in exosomes; (C) Representative Western blot images of CD63, TSG101, and Calnexin proteins in G-MDSCs-derived exosomes; (D) The mean fluorescence intensity of CD63, TSG101, and Calnexin in G-MDSCs-derived exosomes; (E) Representative Western blot images of CD63, TSG101, and Calnexin proteins in exosomes from normal lung tissues derived G-MDSCs culture medium or lung cancer tissues derived G-MDSCs culture medium; (F) Fluorescently-labeled exosomes entered into lung cancer cells. Representative images were filmed after the tissues were fixed and stained (magnification, 400×); (G) Proliferation rate of LLC cells were analyzed by CCK-8 assays every 24 hours following co-culture with G-MDSCs-derived exosomes for 48 hours; (H) Ki67 level in LLC cells were analyzed using RT-qPCR following co-culture with G-MDSC-derived exosomes for 48 hours; (I) Tumor growth rate of lung cancer xenograft with or without adoptive transfer of G-MDSC-derived exosomes; (J) Kaplan–Meier overall survival curve in mice with or without treatment with G-MDSC-derived exosomes. Mean±SEM, ***P<0.005, ****P<0.001.

Fig 5: CD44 translocation to the nucleus and delayed ‘targeted nuclear degranulation’ mediated by CD44/ERM/F-actin. (A) Neutrophils were fluorescently stained for nuclear membrane, cell membrane and CD63, respectively. After stimulation of neutrophils with PMA (100 nM), CD63 co-localization with the cell membrane (15 min) was earlier than CD63 co-localization with the nuclear membrane (20 min) (scale bar: 5 µm). (B) PMA (100 nM) stimulates neutrophils for 0–20 min. Immunofluorescence staining of nuclear membrane (LaminB1,blue), skeleton (F-actin, red) and CD44 (green), respectively. (C) PMA (100 nM) stimulates neutrophils for 0–20 min. WB for changes in nuclear membrane CD44, ERM and CD43 content changes. ERM phosphorylation inhibitor (P-ERMi, 10 µM, 30 min) pre-incubation of neutrophils for 30 min. WB detection of intracellular MPO, histone (H3) changes. (D) WB and ELISA for changes in intranuclear, cytoplasmic and extracellular CD44 content, flow cytometry for changes in cytosolic CD44 content. (E and F) P-ERMi (10 µM, 30 min) pre-incubation of neutrophils. (E) PMA (100 nM) stimulates neutrophils for 30 min, immunofluorescence staining for nuclear (DAPI,blue), P-ERM (green) and CD44 (red). (F) PMA (100 nM) stimulates neutrophils for 120 min, SYTOX fluorescence staining. (G) Importini pre-incubation of neutrophils, immunofluorescence staining of nuclear membrane (LaminB1,blue), skeleton (F-actin, red) and CD44 (green). (H) Importini (30 µM) pre-incubation of neutrophils, WB detection of nuclear membrane CD44 and changes in nuclear MPO content