Fig 2: Experimental workflow for characterization of lymphatic sEVs and proteomic analysis.A, lymphatic fluid was used to isolate sEV particles through size-exclusion chromatography (qEV1 70 nm column; IZON), followed by concentration using a cutoff column (30 kDa). The sEVs were characterized using nanoparticle tracking analysis (NTA) performed in triplicate. B, transmission electron microscopy (TEM) analysis was conducted to examine the size and morphology of lymphatic sEV. C, a dot plot indicates the average size of sEV from each sample in control, melanoma and postoperative lymphatic fluid. D, Western blot analysis was performed to assess the presence of classical sEV markers, including Alix, CD9, and syntenin-1, as well as negative makers, albumin and calnexin in plasma, lymph, and exosomal particles. E, the TMTpro workflow involved labeling lymphatic sEVs collected from afferent channel control, melanoma, and postoperative samples with 18 different TMTpro tags, which were further pooled, cleaned, and then analyzed using mass spectrometry. sEV, small extracellular vesicle; TMT, tandem mass tag.

Fig 3: Chymase modulates integrin and syntenin-1-associated signalling pathways in HLFs. (A-B) AmpliSeq transcriptome analysis illustrating the gene expression levels of various heparan sulphate proteoglycans (HSPGs), including glypicans (GPC) and syndecans (SDC), as well as syntenin-1 (SDCBP), in primary HLFs. ***P < 0.001. (C-D) Effect of chymase treatment on fibronectin (FN), integrin αV (ITGAV) and integrin β3 (ITGB3), phosphorylation of FAK (Tyr397), phosphorylation of Src family kinases (Tyr416), and Akt (Ser473). GAPDH served as a loading control. Representative blots from at least three independent experiments are shown

Fig 4: Quantification of EVs isolated by ultracentrifugation, size exclusion chromatography, or EE affinity column. EVs were isolated from 4, 2, and 1 mL of acellular BAL by SEC, UC, and EE. Preparations were then analyzed for (A) particle yield by Nanosight (n = 3 subjects); (B) protein concentration (n = 3 subjects); (C) assessment of EV transmembrane proteins tetraspanin and syntenin-1 by western blotting; (D) Quantitation of western blot bands (n = 3 subjects); (E) Further confirmation of EV isolation was performed by detection of the intracellular EV protein TSG101. EVs: Extracellular vesicles; BAL: bronchoalveolar lavage; SEC: size exclusion chromatography; UC: ultracentrifugation; EE: exoEasy; CD: cluster of differentiation; Syn-1: syntenin-1; TSG101: tumor susceptibility gene 101.