Melanoma-derived small extracellular vesicles induce lymphangiogenesis and metastasis through an NGFR-dependent mechanism

Abstract

Secreted extracellular vesicles (EVs) influence the tumor microenvironment and promote distal metastasis. Here, we analyzed the involvement of melanoma-secreted EVs in lymph node pre-metastatic niche formation in murine models. We found that small EVs (sEVs) derived from metastatic melanoma cell lines were enriched in nerve growth factor receptor (NGFR, p75NTR), spread through the lymphatic system and were taken up by lymphatic endothelial cells, reinforcing lymph node metastasis. Remarkably, sEVs enhanced lymphangiogenesis and tumor cell adhesion by inducing ERK kinase, nuclear factor (NF)-κB activation and intracellular adhesion molecule (ICAM)-1 expression in lymphatic endothelial cells. Importantly, ablation or inhibition of NGFR in sEVs reversed the lymphangiogenic phenotype, decreased lymph node metastasis and extended survival in pre-clinical models. Furthermore, NGFR expression was augmented in human lymph node metastases relative to that in matched primary tumors, and the frequency of NGFR⁺ metastatic melanoma cells in lymph nodes correlated with patient survival. In summary, we found that NGFR is secreted in melanoma-derived sEVs, reinforcing lymph node pre-metastatic niche formation and metastasis.

Keywords: CD271; NGFR; cell adhesion; lymph node metastasis; lymphangiogenesis; melanoma metastasis; metastasis mechanisms; p75NTR; pre-metastatic niche formation; small extracellular vesicles.

Extended Data Fig. 1. Biophysical properties and cargo of melanoma-derived sEVs.

a, Analysis of sEV (ALIX, CD81 and CD9) and non-sEV markers (GM130 and CALNEXIN) in sEVs from the indicated mouse melanoma cell lines purified by ultracentrifugation. Two independent experiments were performed (n = 2 samples per group). b, Electron microscopy images of the indicated sEVs after iodixanol density gradient. Two independent experiments were performed (n = 2 samples per group). Scale bar, 200 μm. c, Analysis of sEV markers in iodixanol density gradient fractions obtained for B16F10 and B16-F1R2 sEVs. EEA-1 was included as non-sEV marker. Two independent experiments were performed (n = 2 samples per group). d,e, Measurement of the average and mode diameter size and protein content of sEVs purified by ultracentrifugation from the indicated cell lines. Two independent experiments were performed (n = 6 samples per group) f, Z-potential measurements of B16-F1, B16-F10 and B16-F1R2-derived sEVs. N = 2 samples per group. g, Venn diagram for significant upregulated proteins in B16-F10 and B16-F1R2-secreted sEVs compared to B16-F1-derived sEVs. Statistically significant changes were defined using a Student’s t test (FDR < 5%, p value < 0.05). Fold change was set at 1.32 (log2). h, Significantly enriched pathways associated with the upregulated proteins found in B16-F10 and B16-F1R2-secreted sEVs compared to B16-F1-derived sEVs. The enrichment analysis was performed using ClueGO pluggin and used a two-sided test for p value calculation followed by Bonferroni step down correction for p adjusted value. Padj, p adjusted value. i, Quantification of integrins detected by mass spectrometry in the indicated sEVs (n = 2 samples per group). j-l, Schemes showing the experimental planning for LN education experiments. j, Doses of 5 μg of melanoma-derived sEVs were injected intra-footpad every 2 days for the indicated days. At indicated time points, 50,000 B16-F1mCherry or B16-F1-GFP cells were injected intra-footpad (j, k) or in the flank (l). Data represent mean ± s.e.m. and p values were calculated by two-sided Kruskal-Wallis test in d and by one-way ANOVA in e and f.

Extended Data Fig. 2. Analysis of sEV distribution in LNs.

a, Representative images of colocalization of labeled B16-F1R2-secreted sEVs with LYVE-1⁺ LECs and CD169⁺ macrophages in popliteal LNs. Nodules were analyzed 16 h after intra-footpad injection of DiD-labeled sEVs. Two experiments were performed (n = 4 mice per group). Scale bar, 20 μm. b, Gating strategy for the analysis of sEV uptake by LN populations. c, sEV-associated fluorescence in the indicated CD45⁻ cell populations in popliteal and inguinal LNs 16 h after intra-footpad injection of DiD-labeled B16-F10-secreted sEVs determined by flow cytometry. Three experiments were performed (n = 3 samples per group). BECs, blood endothelial cells; FRCs, fibroblastic reticular cells. d, Representative plots showing sEV-associated fluorescence in the indicated LN macrophage populations treated in the same conditions as in (c). Three experiments were performed (n = 3 samples per group). e, Percentage of LECs and CD169⁺F4/80⁺ macrophages exhibiting sEV-associated fluorescence at the indicated times after intra-footpad injection of B16-F1R2 sEVs as determined by flow cytometry. Three experiments were performed (n = 3 samples per group). Data represent mean ± s.e.m and p values were calculated by two-way ANOVA.

Extended Data Fig. 3. Characterization of SK-MEL-147-derived sEVs.

a, Representative profile of nanoparticle size distribution analyzed by nanotracking analysis (NTA) of SK-MEL-147-derived sEVs. Three experiments were performed (n = 3 samples per group). b, Electron microscopy images of SK-MEL-147-secreted sEVs after iodixanol density gradient. Two experiments were performed (n = 2 samples per group). Scale bar, 200 μm. c, Analysis of sEV markers (ALIX, CD63 and CD81) and non-sEV markers (GM130 and CALNEXIN) in SK-MEL-147-derived sEVs purified by ultracentrifugation compared to whole SK-MEL-147 cell extracts. Two experiments were performed (n = 2 samples per group).d, Analysis of sEV markers in the sequential fractions obtained after iodixanol density gradient of a representative SK-MEL-147-derived sEV preparation. EEA-1 was included as non-sEV marker. Two experiments were performed (n = 2 samples per group).

Extended Data Fig. 4. Human LECs incorporate melanoma-secreted sEVs.

a, Representative bright field image of HLECs growing in monolayer, images were obtained from three independent experiments (n = 3 samples). Scale bar, 100 μm. b, Co-staining of lymphatic markers PROX-1 and CD31 in HLECs cultures (n = 3 samples). Scale bar 150 μm .c, Representative immunofluorescence (left panel) and bright field (right panel) images of cultured HLECs exposed to CSFE-labeled SK-MEL-147 sEVs for 16 h. Data were collected from two independent experiments (n = 4 samples per group). Scale bar, 100 μm. d, Representative flow cytometry plots showing apoptotic cell levels in HLECs treated with SK-MEL-147 sEVs for 48 h. Numbers on the gates show the percentage of live cells in each condition (n = 2 samples per group). e, Representative immunofluorescence and bright field images of cultures of HLECs 8 h after exposure to CSFE-labeled SK-MEL-147- or human primary melanocytes (Melano)-derived sEVs. Two experiments were performed (n = 3 samples per group). Scale bar, 100 μm. f, Representative flow cytometry plots for in vitro sEV uptake. Human LECs were treated with PKH67-labeled SK-MEL-147 or melanocyte sEVs and fluorescence was measured at the indicated time points (n = 2 samples per group). g, Median fluorescence intensity (MFI) signal obtained by flow cytometry analysis from measurements performed in (e), (n = 2 samples per group). h, i, Modal distribution and quantification of mean fluorescence intensity (MFI) of sEV-associated fluorescence in HLECs upon exposure to DiD-labeled SK-MEL-147 sEVs in combination with 10 μg/mL GRGDSP or 0.1 μg/mL anti-integrin α_v antibody for 16 h obtained by flow cytometry. Control condition (CTRL) represented HLECs treated with the DiD dye alone. Data were collected from two independent experiments (CTRL, sEVs and sEVs+anti-α_v groups, n = 4 samples, sEVs+GRGDSP group, n= 3 samples). Data represent mean ± s.e.m and p values were calculated by one-way ANOVA.

Extended Data Fig. 5. Melanoma sEVs promote transcriptional changes in LECs.

a, Single sample GSEA analysis showing KEGG significantly downregulated signatures obtained by RNAseq analysis in human LECs upon exposure to SK-MEL-147-derived sEVs during 48 h (n = 3 samples per group). b, GSEA plots of neural-related gene signatures exhibiting significant enrichment in sEV-treated LECs versus non treated cells according to RNAseq data. Nominal p value <0.0001. c, mRNA levels analyzed by qPCR of some of the most up-regulated genes obtained by RNAseq in hLECs. Cells were incubated with SK-MEL-147-derived sEVs (sEVs) or PBS for 24 h and 48 h or with sEV-depleted conditioned medium (CM) from SK-MEL-147 cells for 24 h. Data were collected from two independent experiments (n = 5 independent cell cultures per group) d, GSEAs showing positive enrichment of adhesion-related signatures in sEV-treated LECs versus non treated cells. Nominal p value <0.01. e,f, ICAM-1 expression in HLECs treated SK-MEL-147 sEVs during 48 h. Two experiments were performed (n = 4 samples per group). Scale bar, 25 μm. g,h Quantification and representative images of tumor adhesion on HLECs monolayer. HLECs were pre-treated with SK-MEL-147. sEVs during 48 and subsequently incubated with 5,263 tumor cells/cm² for 3 h before fixation. Two independent experiments were performed (n = 9 samples per group). Scale bar, 50 μm. Data represent mean ± s.e.m and p values were calculated by one-way ANOVA in c and by two-tailed Student t-test in f and g.

Extended Data Fig. 6. Melanoma-derived sEVs promote lymphangiogenesis.

a, GSEA plots for lymphangiogenesis and angiogenesis-related gene sets positively enriched in HLECs treated with SK-MEL-147-derived sEVs for 48 h compared to control LECs (n = 3 samples per group). Nominal p value <0.01. b, Number and length of branches/tubes in endothelial cell tube assays in HLECs incubated for 48 h with SK-MEL-147 sEVs and subsequently plated on matrigel for 16 h. Data were collected from two independent experiments (n = 7 samples per group). c,d, Representative images and quantification of endothelial cell tube assays performed in HMVECs treated in the same conditions as described in (b). Data were collected from two independent experiments (n = 5 samples per group) Scale bar, 100 μm. e,f, Representative images and quantification of tubular structures in co-cultures of HMVECs (CD31⁺ cells) and HLF fibroblasts treated for 48 h with SK-MEL-147 sEVs and untreated (PBS) control. Two independent experiments were performed (CTRL group, n = 6 samples, SK-MEL-147 sEVs group, n = 8 samples). Scale bar, 50 μm. g, Quantitative PCR analysis of LYVE-1 and Prox-1 genes in sorted cell populations from popliteal LNs of Prox-1-tdTomato mice 24 h after injection of DiD-labeled B16-F10 sEVs or control dye (n = 4 samples per group). h,i, Representative histological images of popliteal LNs stained with LYVE-1 (magenta) and Ki67 (brown) and corresponding quantification of Ki67⁺Lyve⁺ cells. Animals were injected intra-footpad 3 times with B16-F1-R2 sEVs for 1 week (n = 5 mice per group, CTRL, n = 8 LN sections and R2 sEVs 10 LN sections). Scale bar, 150 μm. Boxes and whiskers in the box plots in b and d are defined as in Fig. 1. Data represent mean ± s.e.m and p values were calculated by two-tailed Student t-test in b, d and g or by two-tailed Student t test with Welch’s correction in f and i.

Extended Data Fig. 7. sEVs induce macrophage-independent changes in LECs.

a, Expression of pro-lymph-angiogenic genes in sorted CD169⁺F4/80⁺ macrophages. LNs were harvested 48h after intra-footpad injection of B16-F1-R2 sEVs. Data were collected from two independent experiments (n = 4 mice per group). b, Percentage of LECs with DiD-sEV associated fluorescence analyzed by flow cytometry. WT or CD169^DTR mice were treated with diphtheria toxin and 48 h later, B16-F1-R2 sEVs were injected intra-footpad. Animals were sacrificed 24 h later. (n = 5 mice per group). c, Measurement of proliferating PROX-1⁺ LECs in popliteal LNs from WT and CD169^DTR mice. Animals were treated with diphtheria toxin and 24h later, B16-F1-R2 sEVs were injected intra-footpad. Mice were sacrificed 48 h later (n = 5 mice per group). d, Comparison of NGFR mRNA levels (z-score) in cell lines from different tumor types obtained from the CCLE database (melanoma, n = 58; endometrium cancer, n = 25; breast cancer, n = 56; ovary cancer, n = 44; pancreatic cancer n = 42; lung cancer, n = 166 ; central nervous system (CNS) cancer, n = 47; and stomach cancer, n = 34). e, NGFR mRNA levels in a panel of primary melanocytes (Melano1 and Melano2) and human melanoma cell lines. Data were acquired from two independent experiments (n = 4 samples per group). f, Ngfr mRNA levels in mouse melanoma B16 cell lines. Data were collected from two independent experiments (all groups, n = 4 independent cell cultures per group. g, Ngfr fluorescence distribution analyzed by flow cytometry in mouse melanoma sEVs. Plot shows a representative analysis of two independent experiments (n = 2 samples per group). h, NGFR protein levels in human melanoma cell lines compared to primary melanocytes analyzed from a published mass spectrometry data set. (Melano and SK-MEL-147 groups, n = 4 samples, WM-164 and SK-MEL-28 groups, n = 3). Data represent mean ± s.e.m and p values were calculated by two-tailed Student t-test in a and by one-way ANOVA in b-f and h.

Extended Data Fig. 8. NGFR knock-down in metastatic melanoma cell lines.

a, Representative WB showing NGFR protein levels in control (shC) or NGFR shRNA (shNGFR) SK-MEL-147 whole cell lysates and secreted sEVs. Two independent experiments were performed (n = 2 samples per group). b, NGFR protein levels in whole cell lysates and sEVs from control (CTRL) and Ngfr KO B16-F1R2 cells. Two independent experiments were performed (n = 2 samples per group). c, Measurement by nanoparticle tracking analysis (NTA) of the number of particles (right plot) and protein content (left plot) after paired purification of sEVs from the indicated B16-F1R2 cell lines. Data were collected from six independent experiments (n = 6 samples per group). d, Percentage of LN cell types incorporating DiD-labeled sEVs 16 h after footpad injection of control (CTRL) and Ngfr KO B16-F1R2-derived sEVs (n = 3 mice per group). e, Overrepresented pathways associated with the significantly downregulated proteins found in shNGFR SK-MEL-147-derived sEVs compared to shC SK-MEL-147-derived sEVs (n = 3 samples per group). Pathways were obtained using PANTHER software overrepresentation tool applying Fisher’s exact test and FDR for multiple comparison correction. Padj,adjusted p value. f, Representative immunoblotting displaying ERK1/2 phosphorylation levels in HLECs treated with 5 μg/mL of SK-MEL-147-derived sEVs for the indicated times in the presence or absence of 1 μM MEK inhibitor PD0325901. Total ERK-1/2 levels are shown as loading control. Two independent experiments were performed (n = 2 cell samples per group). g, Quantification of phospho-AKT and phospho-mTOR staining in HLECs in basal conditions or after the addition of SK-MEL-147 sEVs for 30 min. Fluorescent signal was measured using Opera high content screening system. (Phosho-AKT fluorescence, CTRL cells n = 3598, sEV n = 3457. Phospho mTOR fluorescence, CTRL n = 517, sEV n =1509). Data represent mean ± s.e.m and p values were calculated by two-tailed paired Student t-test in c, by one-way ANOVA in d and by two-tailed unpaired Student t-test in g.

Extended Data Fig. 9. NGFR promotes tumor adhesion and lymphangiogenesis in LECs.

a,b, Images and quantification of SK-MEL-147-GFP tumor cells attached to a monolayer of human LECs after 3 h incubation. hLECs were pre-treated for 48 h with control shRNA (shC) or NGFR shRNA (shNGFR) SK-MEL-147 sEVs. Treatment with primary melanocytes-derived sEVs (Melano sEVs) was included as additional control. Two independent experiments were performed (n = 14 samples per group). Scale bar, 50 μm. c, NGFR protein levels in hLECs treated with control (shC) or NGFR shRNA (shNGFR) SK-MEL-147 sEVs for 24 h and 48 h. When indicated, cells were treated with THX-B 2 h before harvesting. Two independent experiments were performed (n = 2 samples per group). d,e, Images and quantification of NGFR staining in HLECs incubated with shControl or shNGFR SK-MEL-147 sEVs in the presence or absence of THX-B. Data were acquired from two independent experiments (n = 14 images per group). Scale bar, 25 μm. f, Expression of NGFR and pro-lymphangiogenic genes in HLECs treated with shControl or shNGFR SK-MEL-147 sEVs for 48 h in the presence or absence of JSH-23. Two independent experiments were performed (n = 6 samples per group). g, Quantification of tube structures in endothelial cell tube assays performed in HLECs incubated for 48 h with SK-MEL-147 sEVs alone or in combination with Pro-NGF (n = 5 samples per group). h, Quantification of LYVE-1⁺ cells in histological sections of matrigel plugs 15 days after implantation. SK-MEL-147-derived sEVs were embedded alone or in combination with BDNF or Pro-NGF in matrigel immediately prior to implantation (n = 3 plugs per group). i, Expression levels of neurotrophin receptors and NGFR ligands in popliteal LN exposed to B16-R2 sEVs for 48 h (n = 3 mice per group). Boxes and whiskers in the box plots in g are defined as in Fig. 1. All other data represent mean ± s.e.m and p values were calculated by one-way ANOVA in b-h and two-way ANOVA in i.

Extended Data Fig. 10. NGFR influences LN metastasis

a, Growth curves of flank tumors in mice injected with 200,000 control or Ngfr KO B16-F1R2 cells (n = 6 mice per group). b, HMB-45 histological staining in inguinal LN sections in mice from experiment described in (a) (n = 6 mice per group). Scale bar 150 μm. c, Metastatic foci in inguinal LN sections of animals from experiment described in (a) (n = 6 mice per group). d, Metastatic lesions in LN of mice 15 days after intra-footpad injection of 50,000 control or NGFR KO B16-F1R2 cells (n = 6 mice per group). e, NGFR mRNA levels in primary tumors (PT) and metastatic tumors (Met) according to TCGA melanoma data set (PT group, n = 103 patients and Met group, n= 367 patients). f, NGFR mRNA levels in primary tumors (PT LN⁺) and metastatic tumors (Met LN⁺) with LN involvement. (PT LN⁺ group, n = 42 patients and Met LN⁺ group, n = 171 patients). Data represent mean ± s.e.m and p values were calculated by two-way ANOVA in a, by two-tailed Student t-test in c and d and by two-sided Mann-Whitney test in e and f.

Figure 1.. Melanoma-secreted sEVs are retained through the lymphatic system.

a, Representative images of sEV-associated signal in mice injected intra-footpad 3 weeks with the indicated NIR815-labeled sEVs (n = 2 mice per group). Po, popliteal LN; Il, Iliac LN; In, inguinal LN; Li, liver. b,c, Representative images and quantification of NIR815-associated signal in LNs from mice treated as above. Two independent experiments were performed (n = 4 LNs per group). L, lateral LN; CL, contralateral LN. d,e, Representative images and quantification of sEV-associated signal in mice 1, 4, 24 and 48 hours after footpad injection with NIR815-labeled sEVs. Data correspond to two independent experiments (n = 4 mice per group). Squares indicate popliteal LN area. f, Representative images of the distribution of melanoma DiD-labeled sEVs in popliteal LNs 16 h after intra-footpad injection (n = 4 LNs per group). Scale bar, 150 μm. g,h, Representative flow cytometry plots and quantification of B16-F1-GFP⁺ cells within the CD45⁻ population in LNs educated with sEVs for 10 days. LN were analyzed 24 h post-injection of tumor cells (PBS and R2 n = 5 mice per group, F1 and F10 n = 4 mice per group). i,j, Representative images and quantification of B16-F1-mCherry⁺ cells in sections of popliteal LNs 10 days post tumor cell injection. Melanoma-derived sEVs or PBS were injected intra-footpad for 10 days prior to tumor inoculation. Two independent experiments were performed (CTRL, F1, F1R2 groups, n = 5 mice per group, F10 group, n = 4 mice). Scale bar, 20 μm. k,I, Representative images and quantification of metastatic area in inguinal LNs of animals bearing B16-F1-GFP flank tumors and educated with B16-F1-R2 sEVs injected intra-footpad for 21 days. Two independent experiments were performed (n = 12 mice per group). Scale bars, 500 μm and 200 μm. Boxes in the box plots in h and j define the IQR split by the median, with whiskers extending to the most extreme values within 1.5 × IQR beyond the box. All other data represent mean ± s.e.m. and p values were calculated by two-way ANOVA in c and e, by one-way ANOVA in h and j and by two-tailed Student’s t test in l.

Figure 2.. Small EVs are incorporated by LN stromal and immune cells.

a, Representative images of earlobe lymphatic vessels in Prox-1-GFP mice 1 h after intra-ear injection of PKH26-labeled B16-F10 sEVs. Top right inset displays the orthogonal projection corresponding to the white dashed line (n = 2 mice per group). Scale bars, 100 μm and 20 μm. b, Representative images of cervical LNs 16 h after intra-footpad injection of PKH26-labeled B16-F1 and B16-F10 sEVs. Arrows indicate areas of overlapping PKH26 and LYVE-1 staining (n = 3 mice per group). Scale bar, 200 μm and 20 μm. c, Representative images of two consecutive popliteal LN sections dissected 16 h after intra-footpad injection of DiD-labeled B16-F1-R2 sEVs. White lines delineate areas of lymphatic (upper panels) and macrophage (lower panels) stained networks (n = 3 LNs per group). Scale bar, 250 μm and 50 μm. d, e, Representative flow cytometry plots and quantification related to B16-F10 sEV uptake in the indicated stromal and immune populations. DiD-labeled sEVs were injected intra-footpad and LNs populations were analyzed 16 h later. Gates were depicted based on corresponding dye-only signal for each population. Gating strategy is described in Extended Data Fig 2b. Three independent experiments were performed (n = 4 LNs analyzed). BECs, blood endothelial cells; FRCs, fibroblastic reticular cells; DC, dendritic cells. f, Representative plots of B16-F1R2 sEV-associated fluorescence in LECs and CD169⁺F4/80⁺ macrophages 4 h after intra-footpad injection. Three independent experiments were performed (n = 3 LNs analyzed). g, Median DiD fluorescence intensity associated to B16-F1-R2 sEVs in LECs and CD169⁺F4/80⁺ macrophages (mos) at the indicated times. Two independent experiments were performed (n = 3 LNs analyzed). All data represent mean ± s.e.m. and p values were calculated by two-tailed Student’s t test in e and by two-way ANOVA in g.

Figure 3.. Melanoma-derived sEVs influence LEC transcriptional profile and promote adhesion.

a, Single sample GSEA analysis showing significantly enriched KEGG signatures in human LECs treated with SK-MEL-147 sEVs or PBS for 48 h (n = 3 samples per group). b, Correlation between RNAseq data in LECs and proteomic data in SK-MEL-147 sEVs. Color code indicates significantly regulated gene-protein pairs (FDR < 5%). c, Top pathways significantly enriched in the group of gene-proteins positively correlated shown in (b). Pathways were obtained using PANTHER overrepresentation analysis applying Fisher’s exact test and FDR correction. d,e, Representative images of PKH26-labelled SK-MEL-147 cells adhered to sEV-treated LECs in flow. LECs were previously exposed to PKH67-labeled sEVs from primary melanocytes (Melano) or SK-MEL-147 cells during 24 h. Plot in (e) shows quantification of attached tumor cells at t = 4 h. Two independent experiments performed (all groups, n = 20 fields from one representative experiment. Scale bar, 50 μm. f, ICAM-1 expression in hLECs treated with SK-MEL-147 sEVs for 48 h. Two independent experiments performed (n = 6 LNs per group). g,h, Representative images and quantification of ICAM-1 expression in LNs treated with B16-F1-R2 sEVs intra-footpad for 10 days. Two independent experiments were performed (n = 7 LNs per group). Scale bar, 100 μm and 200 μm. i, LYVE-1 and ICAM-1 staining in LNs treated with F1-R2 sEVs or PBS for 48h (n = 3 LNs per group). Scale bar, 50 μm. j, Quantification by flow cytometry of LECs expressing high levels of ICAM-1 in LNs of animals injected intra-footpad with B16-F1R2 sEVs for the indicated times. Two independent experiments were performed (CTRL and 48 h, n = 3 LNs per group and 7 days, n = 4 LNs per group). k,l, Representative images and quantification of the adhesion of SK-MEL-147 cells (green) to HLEC monolayers in the presence of Fc ICAM blocking molecule or vehicle. HLECs were previously exposed or not to SK-MEL-147 sEVs. Two independent experiments were performed (n = 4 samples per group). Scale bar, 20 μm. All data represent mean ± s.e.m. and p values were calculated by two-tailed Student’s t test in e and h and by one-way ANOVA in f, j and l.

Figure 4.. Melanoma-sEVs promote LN lymphangiogenesis.

a, Heatmap of angiogenesis and lymphangiogenesis-related genes based on RNAseq data (all groups, n = 3 samples per group b, Expression of lymph/angiogenesis-related genes in SK-MEL-147 sEVs-treated or control hLECs. Data were obtained from 2 independent experiments (n = 6 independent cell cultures per group). c, Representative WB of phospho-VEGFR3 and total VEGFR3 levels in HLECs treated with SK-MEL-147-derived sEVs. Three independent experiments were performed (n = 3 samples per group). d, Representative pictures of endothelial cell tube assays performed in HLECs growing on matrigel for 16 h after treatment with SK-MEL-147-derived sEVs. Data were obtained from 3 independent experiments (n = 15 samples per group). Scale bar, 100 μm. e,f, Representative images and quantification of LYVE-1⁺ cells in control (PBS) and SK-MEL-147 sEV-embedded matrigel plugs. Data were collected from two independent experiments (CTRL, n = 5 plugs and sEVs, n = 4 plugs). Scale bar, 50 μm. g, Representative images of consecutive sections of a sEV-embedded matrigel plugs stained with LYVE-1 and F4/80 (n = 4 plugs per group). Scale bar, 250 μm and 80 μm (inset). h,i, Representative images and quantification of luminescence associated to LNs of Vegfr3-EGFP-luc mice after 7 days of exposure to B16-F10 sEVs. Po, popliteal LN; In, inguinal LN; Ax, axillary LN. Data were obtained from three independent experiments (Popliteal CTRL, axilary CTRL and inguinal sEV groups n = 3 LNs; inguinal CTRL groups, n = 2 LNs; axillary sEVs group, n = 4 LNs; popliteal sEVs group, n = 5 LNs). j,k, Representative images and quantification of LYVE-1 staining in LNs after 48 h of intra-footpad injection with B16-F1-R2 sEVs. Two independent experiments were performed (n = 8 LNs per group). Scale bar 250 μm. l,m, Representative images and quantification of LEC proliferation in LNs after 24 h of intra-footpad injection with B16-F1-R2 sEVs (n = 5 LNs per group). Scale bar 40 μm. All data represent mean ± s.e.m. and p values were calculated by two-tailed Student’s t test in f, k and m and by two-way ANOVA in b and i.

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.

Figure 6.. Melanoma sEVs activate NGFR, MAPK and NF-kB signaling pathways in LECs.

a,b, Representative images and quantification of p65 staining in HLECs after exposure to TNFα or SK-MEL-147-derived sEVs for the indicated times. Arrows indicate cells with p65 nuclear staining (n = 5 samples per group). Scale bar, 50 μm. c,d, Representative confocal images and quantification of p65 staining in HLECs in basal conditions or after the addition of TNFα, control shRNA or NGFR shRNA SK-MEL-147 sEVs for 30 min. p65 translocation was analyzed using a confocal high content screening system. Plot shows data from one representative experiment out of two (CTRL, n = 5325 cells, CTRL shC, n = 3355 cells and shNGFR, n = 3005 cells). Scale bar, 40 μm. e-g, Representative confocal images and quantification of nuclear area (f) and average fluorescence intensity (g) for p65 staining in HLECS exposed to of SK-MEL-147-derived sEVs in the presence or absence of NF-kB inhibitor JSH-23 or NGFR inhibitor THX-B. Data were collected from two independent experiments (n = 12 samples per group except THX-B and JSH-23 groups, n = 7 samples for f and n = 6 samples for g). h,i, Representative WB of ERK1/2 phosphorylation levels in HLECs treated with shControl (shC) and shNGFR SK-MEL-147-derived sEVs for the indicated times. Data were collected from four independent experiments (n = 4 samples per group). j,k, Representative confocal images and quantification of phospho-ERK1/2 in HLECs in basal conditions or after the addition of shControl (shC) or shNGFR SK-MEL-147-derived sEVs for 30 min. Phospho-ERK1/2-associated cell fluorescence was analyzed using a confocal high content screening system. Plot shows data from one representative experiment out of two (CTRL, n = 4777 cells, CTRL shC, n = 3419 cells and shNGFR, n = 2323 cells). Scale bar, 50 μm. All data represent mean ± s.e.m. and p values were calculated by one-way ANOVA except by two-way ANOVA in i.

Figure 7.. sEVs induced ICAM-1 expression and lymphangiogenesis through NGFR pathway.

a, Quantification of ICAM-1 fluorescence area in HLECs treated with shControl (shC) or shNGFR SK-MEL-147-derived sEVs for 48 h with or without THX-B. Data were collected from three independent experiments (CTRL, n = 36, THX-B, n = 26, shC sEVs and shC sEVs+THX-B, n = 35, shNGFR sEVs, n = 18 fields per group). b,c, Representative images and quantification of ICAM-1 fluorescence of HLECs treated with SK-MEL-147-derived sEVs with or without MEK inhibitor or JSH-23. Data were collected from two independent experiments (n = 18 fields per group). Scale bar, 40 μm. d,e, Representative images and quantification of endothelial tubbing assays performed with HLECs previously exposed to shControl (shC) or shNGFR SK-MEL-147-derived sEVs for 48 h with or without THX-B, JSH-23 or MEK inhibitors (CTRL and shC sEVs groups, n = 14 samples and rest of groups, n = 7 samples per group). Scale bar, 100 μm. f,g, Representative images and quantification of LYVE-1⁺ cells in PBS, shControl (shC) or shNGFR sEVs-embedded matrigel plugs 15 days post-injection. Scale bar, μm 150. Two independent experiments were performed (CTRL and shC sEVs groups, n = 10 plugs analyzed and shNGFR sEVs group, n = 9 plugs analyzed). h,i, Quantification of LYVE-1 area and representative images of popliteal LNs from mice educated with intra-footpad injections of B16-F1R2 Ngfr KO or control (CTRL) sEVs for 13 days. (n = 8 LNs analyzed). Scale bar, 40 μm. j,k, Representative LYVE-1 histological staining in inguinal LNs of mice with B16-F1R2 flank tumors and corresponding quantification. Animals were treated or not intraperitoneally with 2.5 mg/Kg of THX-B twice a week, starting on day 7 post-injection of tumor cells. Animals were sacrificed at 21 days (n = 16 LNs analyzed). Scale bar, 200 μm. All data represent mean ± s.e.m. and p values were calculated by one-way ANOVA in a- g and by two-tailed Student’s t test in h and k.

Figure 8.. EV-shed NGFR favors LN metastasis and influences survival.

a,b, Representative images and quantification of B16-F1-mCherry⁺ cells in popliteal LNs. Mice were educated with B16-F1-GFP and B16-F1-NGFR-GFP sEVs as in Fig. 1i. Data from two independent experiments (n =10 mice per group, GFP group 28 LN sections and NGFR-GFP group 26 LN sections). Scale bar, 200 μm and 40 μm. c,d, Representative images and quantification of B16-F1-mCherry⁺ cells in popliteal LNs. Animals were educated with control or Ngfr KO B16-F1R2 sEVs as in Fig. 1i (n = 5 mice per group; CTRL group, n = 21 LN sections and Ngfr KO group, n = 24 LN sections). Scale bar, 20 μm. e, Metastatic area in mice educated with control and Ngfr KO B16-F1R2 sEVs as described in Fig. 1k. Two independent experiments were performed (n = 9 mice per group). f, Survival of animals educated with control and Ngfr KO B16-F1R2-secreted sEVs as indicated in (e) (CTRL sEV, n = 12 mice and Ngfr KO sEV, n = 10 mice). g-i, Percentage of animals (g) number of LN metastases (h) and representative images (i) in animals bearing B16F1-R2 flank tumors treated or not with THX-B as in Figure 7j (vehicle n = 7 mice and THX-B n = 8 mice). j, NGFR h Score in skin and LN sections from melanoma patients (n = 26 skin/soft tissue samples and n = 17 LN samples). k, Percentage of NGFR⁺MITF⁺ tumor cells in skin and LN samples from melanoma patients (n = 21 matched samples). l, Overall survival (OS) of stage II/III melanoma patients according to NGFR⁺MITF⁺ cell number in LN biopsies (less than 75 NGFR⁺MITF⁺ cells, n = 13 patients, more than 75 NGFR⁺MITF⁺ cells, n = 12 patients). Boxes and whiskers in the box plots in b and d are defined as in Fig. 1). All other data represent mean ± s.e.m f, k and l. and p values were calculated by two-tailed Student’s t test with Welch’s correction b, d and e, by two-sided Mann-Whitney test in j, by two-sided paired Wilcoxon matched-pairs signed rank test in k and two-sided log rank test in f and l.