Journal: Journal of Extracellular Vesicles

Article Title: Tumour extracellular vesicles induce neutrophil extracellular traps to promote lymph node metastasis

doi: 10.1002/jev2.12341

Figure Lengend Snippet: EVs are essential for LNs NETs formation and metastasis . (a) Statistical analysis of TCGA mRNA data of EV synthesis related genes in primary esophageal adenocarcinoma (EAD) tumour tissue. n = 15 (Stage I‐IIA), n = 65 (Stage IIb‐IV). (b) Kaplan‐Meier survival curves comparing the survival of EAD patients with low versus high levels of VAMP7 mRNA in tumour. (c) Schematic illustration of EV pre‐treatment in animal models. (d) Representative images of tumour draining LNs on day 14 post tumour inoculation for B16F10, with or without pre‐treatment of EVs Scale bars represent 100μm. (e) Quantification of the area of neutrophils (Ly6G), NETs (H3Cit) and tumour (GFP) in day 14 LN for B16F10, with or without EV pre‐treatment. (f) Representative Western Blot images indicating the knockdown of Rab27a expression in B16F10 cells. (g) Nanoparticle Tracking Analysis (NTA) indicates Rab27a knockdown in B16F10 cells leads to decreased EV secretion. (h) Representative images of tumour draining LNs on day 14 post tumour inoculation for B16F10, comparing mice injected with B16F10 infected with lentivirus containing scramble RNA or Rab27a shRNA. Scale bars represent 100μm. (i) Quantification of the area of neutrophils (Ly6G), NETs (H3Cit) and metastasis (GFP) in day 14 LNs for B16F10, comparing mice injected with B16F10 infected with lentivirus contain scramble RNA or Rab27a shRNA. Each data point is an image analysed, mouse n =10. Data shown as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.005; **** P < 0.001 by Mann–Whitney t test.

Article Snippet: A549, BEAS‐2B and B16F10 cells were purchased from ATCC and were cultured in media with EV‐free FBS (ultracentrifuged for 18 h at 100,000 × g ).

Techniques: Western Blot, Knockdown, Expressing, Injection, Infection, shRNA, MANN-WHITNEY

Journal: Journal of Extracellular Vesicles

Article Title: Tumour extracellular vesicles induce neutrophil extracellular traps to promote lymph node metastasis

doi: 10.1002/jev2.12341

Figure Lengend Snippet: The dynamic of LN neutrophil recruitment and NET deposition in mouse model . (a) Schematic illustration of the animal model construction. 250k H59 or B16F10 cells were injected into the flank of C57bl/6 mice (b) Percentage of LN viable neutrophils within all viable LN cells on a time course by flow cytometry. (c) Representative images of LNs on a time course for both H59 lung cancer and B16F10 melanoma. Scale bars represent 100μm. Zoomed NETs details are shown in bottom right. Scale bars represent 20 μm. (d) Quantification of the area of LN neutrophils (Ly6G), NETs (H3Cit) and tumour (GFP). Each data point is an image analysed, mouse n =10. Data shown as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.005; **** P < 0.001 by one‐way ANOVA.

Article Snippet: A549, BEAS‐2B and B16F10 cells were purchased from ATCC and were cultured in media with EV‐free FBS (ultracentrifuged for 18 h at 100,000 × g ).

Techniques: Animal Model, Injection, Flow Cytometry

Journal: Journal of Extracellular Vesicles

Article Title: Tumour extracellular vesicles induce neutrophil extracellular traps to promote lymph node metastasis

doi: 10.1002/jev2.12341

Figure Lengend Snippet: Neutrophil depletion and NETs inhibition abolishes LN metastasis . (a) Schematic illustration of the treatments in animal models. (b) Flow cytometry on blood leukocyte indicates sufficient depletion of neutrophils. (c) Representative images of tumour draining LNs on day 14 post tumour inoculation for both H59 and B16F10, comparing wildtype/no treatment mice to neutrophil‐depleted, pad4 knockout or NEi treated mice. Scale bars represent 100 μm. (d)–(h) Quantification of the area of neutrophils (Ly6G), NETs (H3Cit) and metastasis (GFP) in fay 14 LNs for both H59 and B16F10, comparing wildtype/no treatment mice to neutrophil‐depleted, pad4 knockout or NEi treated mice. Each data point is an image analysed, mouse n =10. Data shown as mean ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.005; **** P < 0.001 by Mann‐Whitney t test.

Article Snippet: A549, BEAS‐2B and B16F10 cells were purchased from ATCC and were cultured in media with EV‐free FBS (ultracentrifuged for 18 h at 100,000 × g ).

Techniques: Inhibition, Flow Cytometry, Knock-Out, MANN-WHITNEY

Journal: Journal of Extracellular Vesicles

Article Title: Tumour extracellular vesicles induce neutrophil extracellular traps to promote lymph node metastasis

doi: 10.1002/jev2.12341

Figure Lengend Snippet: Lymphatic endothelial cells (LECs) are the LN recipient cells of EVs LECs regulate neutrophil recruitment and NETs formation. (a) Representative images of draining LNs after footpad B16F10 EV injection, showing LECs uptaking EVs Scale bars represent 20μm. (b) Representative images of LECs after A549 and BEAS‐2B EV treatment. Scale bars represent 100μm. (c) Quantification of the EV uptake (relative fluorescence area of CFSE to DAPI) in LECs. (d) Schematic illustration of the Boyden chamber transwell assay. (e) Quantification of Boyden chamber transwell assay, showing neutrophil migration towards CM from treated LECs. Data shown as mean ± SEM. *, P < 0.05; ***, P < 0.005; **** P < 0.001 by Mann–Whitney t test or One‐Way ANOVA. (f) Representative images of draining LNs after footpad PBS or different doses of B16F10 EV injection, indicating the subsequent neutrophil recruitment and NETs deposition. Scale bars represent 500 μm. (g) Representative images of draining LNs after footpad PBS or 10 μg B16F10 EV injection, indicating the increased expression of premetastatic marker VEGFR3. Scale bars represent 50 μm.

Article Snippet: A549, BEAS‐2B and B16F10 cells were purchased from ATCC and were cultured in media with EV‐free FBS (ultracentrifuged for 18 h at 100,000 × g ).

Techniques: Injection, Fluorescence, Boyden Chamber Transwell Assay, Migration, MANN-WHITNEY, Expressing, Marker