Figure 5. Metastatic melanoma-derived sEVs contain NGFR and transfer it to LECs.

a, Representative WB of NGFR protein levels in sEVs from human primary melanocytes and melanoma cell lines (upper panels) or murine cell lines (lower panels). Two independent experiments were performed (n = 2 samples per group). b, Representative overlay flow cytometry plots of NGFR staining on SK-MEL-28 and SK-MEL-147 sEVs. Two independent experiments were performed (n = 2 samples per group). c, NGFR mRNA levels in human LECs treated for 24 h and 48 h with SK-MEL-147-derived sEVs or conditioned medium (CM). Data were obtained from two independent experiments (n = 4 independent cell cultures per group, except 24h sEVs, n = 6 and 48h sEVs and cCM, n = 5). d, Representative images of NGFR staining in HLECs exposed to SK-MEL-147 sEVs for 48 h. Three independent experiments were performed (n = 6 cell culture samples per group). Scale, 30 μm. e, NGFR mRNA levels in human LECs exposed to sEVs from melanocytes or different melanoma cell lines for 48 h. Data were collected from two independent experiments (n = 6 samples per group). f-h, Representative images of LYVE-1 and GFP-expression in LNs 16 h after the injection of B16-F1-NGFR-GFP sEVs. White lines delineate areas of LYVE-1⁺ lymphatic network. Quantification of GFP⁺ area and mean fluorescence in LYVE-1 regions are shown in (g) and (h), 4-3 whole sections per LN were analyzed. Data were obtained from 2 independent experiments (n = 6 LNs per group). Scale bar, 200μm (left images) and 50 μm (right image). i,j, Representative flow cytometry plots and quantification of NGFR⁺ cells in HLECs exposed to SK-MEL-147 sEVs for 4h. Two independent experiments were performed (n = 3 samples per group). All data represent mean ± s.e.m. and p values were calculated by two-tailed Student’s t test in g, h and j and by one-way ANOVA in c and e.