Fig 1: SEVs loaded with a large amount of BST2 contribute to PTC cell growth and lymphangiogenesis.A The proliferation rate of BCPAP cells was downregulated after the knockdown of BST2 (*P < 0.05, **P < 0.01). B The cell migration of BCPAP cells was downregulated after the knockdown of BST2 (***P < 0.001). NC: negative control. C The proliferation rate of BCPAP cells was upregulated after overexpression of BST2 (**P < 0.01, ***P < 0.001, ****P < 0.0001). D The cell migration of BCPAP cells was upregulated after overexpression of BST2 (****P < 0.0001). E, F The morphology and size of BCPAP-derived sEVs were determined by TEM and NTA; scale bar: 100 nm. G SEVs markers CD81, CD63, ALIX, Calnexin, and sEVs BST2 levels were measured using western blot. H Lymph tube formation response to OE-BST2 sEVs is determined (***P < 0.001). I–J The cell migration and proliferation of HLEC cells in response to OE-BST2 sEVs are shown (*P < 0.05, **P < 0.01, ****P < 0.0001). Error bars represent standard deviations.
Fig 2: The correlation of differentially expressed protein levels of BST2 in serum sEVs with PTMC progression.A Venn diagram of differentially expressed proteins from different comparisons. B BST2 levels according to proteomic analysis (n = 10 for NLNM-sEVs and LNM-sEVs; *BN-sEVs vs. other groups, *P < 0.05, ****P < 0.0001; #NLNM-sEVs vs. LNM-sEVs, ##P < 0.01). C Western blot analysis showed BST2 levels in serum sEVs (n = 3 for BN-sEVs, NLNM-sEVs, and LNM-sEVs, *BN-sEVs vs. other groups, **P < 0.01, ***P < 0.001; #NLNM-sEVs vs. LNM-sEVs, #P < 0.05); Flotillin-1 as sEVs protein marker was used as the control. D ELISA results revealed BST2 protein concentrations in serum sEVs of the validation cohort (**P < 0.01, ****P < 0.0001). E ROC curve analysis of the sensitivity, specificity, and AUC of serum sEVs BST2 level in the validation cohort. The T refers to the whole group of PTMC samples (LNM and NLNM). Error bars represent standard deviations.
Fig 3: SEVs BST2 promoted lymph node metastasis of PTMC in vivo.A Representative gross anatomical image of the lower limb of a nude mouse with footpad tumor (left arrow) and metastatic popliteal lymph node (right arrow). B Images of the popliteal lymph nodes in control-sEVs-BCPAP or BST2-sEVs-BCPAP groups (n = 12). C Statistical comparison of the volume of popliteal lymph nodes in each group (n = 12, **P < 0.01). D Representative images of HE staining and BST2 IHC staining for popliteal lymph nodes in each group with different magnifications. Scale bars: 100 μm for the 10 × magnified images and 50 μm for the 20 × magnified images. E The IHC staining was assessed as the sum of the percentage and intensity scores (*P < 0.05). Error bars represent standard deviations.
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