DOI:.38/ncb3399 a b c d FSP DAPI 5mm mm 5mm 5mm e Correspond to melanoma in-situ Figure a DCT FSP- f MITF mm mm MlanaA melanoma in-situ DCT 5mm FSP- mm mm mm mm mm g melanoma in-situ MITF MlanaA mm mm mm h GPNMB FSP mm melanoma in-situ mm MlanaA mm DCT 5mm FSP- 5mm FSP- GPNMB 5mm Supplementary Figure : Fibroblasts aggregation at a distance from melanoma regions contain melanoma particles. (a-d) Immunofluorescence analysis of sections from four melanoma patients. DAPI-stained nuclei appear in blue. (e) Left: Immunohistochemistry staining (X magnification) of MITF and MelanA in in situ melanoma sample. Right: Immunofluorescence analysis (63X magnification) of DCT (green) and HMB-5 (red) or FSP- (green) and HMB-5 (red). (f) Left: H&E staining (X magnification) of in situ melanoma. Right: Immunofluorescence analysis (63X magnification) of HMB-5 (red) and DCT (green) or FSP- (green). Yellow signal indicates co-localization. (g) Left: H&E staining, immunohistochemistry for MITF and MelanA for in situ melanoma (X magnification). Right: Immunofluorescence analysis of GPNMB (red) and FSP- (green). Yellow signal indicates co-localization of FSP- and GPNMB (63X magnification), in dashed square higher magnification image (x magnification). (h) From left to right in in situ melanoma: H&E staining, immunohistochemistry (X magnification) for MelanA, immunofluorescence analysis of HMB-5 (red) and DCT (green), HMB-5 (red) and FSP- (green), GPNMB (red) and FSP- (green). Yellow signal indicates co-localization (63X magnification). WWW.NATURE.COM/NATURECELLBIOLOGY
a Normal b Invasive melanoma c Invasive melanoma DCT DAPI mm MITF mm FSP- GPNMB mm MlanaA mm d Invasive melanoma f CD 68 FSP DAPI mm MlanaA mm e 5mm DCT mm CD 68 FSP DAPI fluorescence intensity GPNMB 5mm 5mm GPNMB fibroblasts fluorescence intensity Supplementary Figure Fibroblasts aggregation at a distance from melanoma regions contain melanoma particles. (a) Normal skin stained for HMB-5 (red) and DCT (green). DAPI-stained nuclei appear in blue (63X magnification). (b) Immunohistochemistry for MITF and MelanA for invasive melanoma (X magnification) shown in Figure. (c) Immunofluorescence analysis of FSP- (green) and GPNMB (red) for invasive melanoma (X magnification). Higher magnification image in the lower panel (X magnification). (d) Left: H&E staining and immunohistochemistry for MelanA (X magnification) for invasive melanoma. Right: Immunofluorescence analysis of DCT (green) and HMB-5 (red) for invasive melanoma (63X magnification). (e) Melanosome quantification method. Immunofluorescence analysis of dermal fibroblasts in superficial melanoma patient samples reveals staining for GPNMB (red) indicative of. Left: Red fluorescence regions within fibroblast cytoplasm at the size of 3-6 nm are marked and were analyzed for fluorescence intensity (n=8 ). Right: Whole cell fibroblasts were marked and analyzed for fluorescence intensity (n= cells). (f) A single representative sample is shown stained for FSP (green) and macrophage marker CD68 (red). White arrow indicates non-specific staining, and yellow signal indicates colocalization of FSP- and CD68. DAPI-stained nuclei appear in blue. WWW.NATURE.COM/NATURECELLBIOLOGY
a Tyrosinase activity MNT fractions f Relative mrna expression in MNT fractions h Number of 3.5.5.5 whole cell WC NADH (ND) short RNA whole WC-short cell mature Mature Early pre-mature b whole mature early pre-mature.e+.e+.e+ 8.E+ 6.E+.E+.E+ y = E+9x - E+9 R² =.9997 cytoplasm pre-mature mature whole cell ladder g Tyrosinase HaCat treatment:.e+ 6 Melanin concentration [µg/ml] Control Melanosomes Number of Rab7a (loading ) Tyrosinase Dapi Tyrp Dapi.E+.E+.E+ 8.E+ 6.E+.E+.E+ µm c M r (K) 7 5 FACS analysis Exosome isolation: µm nm y = 7E+x + 3E+8 R² =.993 µm µm.e+..5..5. Protein concentration [mg/ml] d µm µm CD63 CD8 CD63 CD8 e Tyrosinase µm µm DCT GPNMB CD63 α-enolase HSC7 (loading ) melanosome exosome i h h 36h osteopontin 5µm M r (K) µm µm 7 7 7 5 7 Supplementary Figure 3 Melanoma transfer into fibroblasts leads to reprogramming. (a) Tyrosinase activity of MNT- whole cell extracts or isolated mature and pre-mature. Values are normalized to total protein concentration. Error bars represent ± SEM, indicates p<.5 (n=5 independent experiments). (b) Tyrosinase protein levels in indicated fractions. Rab7a was analyzed as a loading. (c) Electron microscopy analysis of exosomes. (d) Expression of CD63 and CD8 in exosomes purified from MNT- cells by FACS analysis. Upper panel shows the analysis of expression of CD63 (blue) compared to unstained exosomes (red). Middle panel shows the analysis of expression of CD8 (blue) compared to unstained exosomes (red). Lower panel shows the analysis of expression of exosome markers CD63 and CD8 (blue) compared to unstained exosomes (red). (e) Protein levels in isolated from MNT- cell homogenates and exosomes isolated from MNT- conditioned medium. Hsc7 was used as a loading. (f) ND- levels in indicated fractions, normalized to GAPDH (n= independent experiment). (g) Immunofluorescence staining of HaCat cells upon melanosome treatment. Melanosomal markers: tyrosinase, Tyrp, and HMB-5 appear in yellow, red, and green, respectively. DAPI-stained nuclei appear blue. (h) Standard curves of melanosome number based on melanin and protein concentrations. (i) Fibroblasts migration upon osteopontin (nm) treatment. Unprocessed scans of Western Blots are shown in Supplementary Fig. 8. WWW.NATURE.COM/NATURECELLBIOLOGY 3
a b Liposomes /cell melanoma /cell 5µm c Relative proliferation.7.6.5..3.. Melanoma )cell/5( Liposomes )cell/5( days d Relative mrna expression f Uptake 5/cell l i 8 6 Normal (/cell) Melanoma (/cell) IL-β IL-6 IL-8 CXCL CXCL COX liposomes 3nm Particles per ml 7.E+8 6.E+8 5.E+8.E+8 3.E+8.E+8.E+8.E+ 5mm exosomal fraction (7-nm) Relative mrna expression e 3.5.5.5 5mm GPNMB melanosomal fraction (-6nm) NS nm..8.6.. m Relative melanin in melanoma medium Liposomes (5/cell) Melanoma (5/cell) IL-β IL-6 IL-8 CXCL CXCL COX Number of vesicles per fibroblast 7 6 5 3 liposomes % of vesicles from total 9 8 7 6 5 3 Relative melanin level in fibroblasts exosomes 8 6 g fluorescence intensity other 8 6 k h 6 5 3.8x + 5E-y =.97 = ²R 3 number of liposomes CD63 5 37 86 55 Secreted Exosomes MNT- whole cell melanoma medium number of liposome per cell average j melanoma release medium + n nm melanoma medium CD8 Tyrosinase In fibroblasts DAPI 5mm CD63 CD8 µm µm µm µm o melanoma 5µm 5µm p relative melanoma cells proliferation.5.5 release 3 days q (SB9) fibroblasts 5µm r Relative mrna expression 3.5.5.5 IL-β IL-6 IL-8 CXCLCXCL COX Supplementary Figure Melanoma transfer into fibroblasts leads to reprogramming. (a) Dynamic light scattering image of liposomes from a NanoSight analysis. Middle: Snapshot image of liposomes movie (Supplementary video ). (b) Fibroblast migration upon indicated treatments. (c) Growth rate of fibroblasts upon indicated treatments (n= independent experiments). (d,e) The pro-inflammatory gene signature of CAFs upon indicated treatments. Data were normalized to GAPDH (n=3 independent experiments). (f) Left: Representative images of fibroblasts treated with 5 rhodamine-labeled liposomes or (immunostained with GPNMB) per cell. Right: Average number of vesicles per cell (n=6 cells for each group, independent experiments). (g) Standard curve of liposome number. (h) Left: Number of liposomes per cell, calculated based on the standard curve (n=3 measurements each of 9 3 cells). Right: Representative image of fibroblasts treated with 5 rhodamine-labeled liposomes per cell. (i) Left: Electron microscopy analysis of secreted vesicles. Right: Higher magnification of the same sample. Red arrows indicate exosomes. Graphs: Percentages of indicated vesicles (n=3 individual experiments). (j) Electron microscopy analysis of secreted. (k) FACS analysis of CD63 and CD8 expression in indicated vesicles. Unstained MNT- cells appear in pale purple, stained MNT- cells in purple, unstained MNT- exosomes in pale red, stained MNT- exosomes in red, unstained MNT- in pale blue, and stained MNT- in blue. (l) NanoSight analysis of the secreted vesicle population upon P38 treatment (n=3 independent experiments). (m) Left: Melanin levels in melanoma medium upon SB9 treatment. Data were normalized to RNU6B (n= independent experiments). Right: Melanin levels in fibroblasts treated with medium from melanoma cells incubated with or without SB9. Fibroblast medium was used as a. Values were normalized to total protein amount (n=8 samples, 3 independent experiments). (n) Immunostaining of fibroblasts treated with medium from melanoma cells that were pre-treated with SB9. Tyrosinase is stained green; DAPI-stained nuclei appear in blue. (o) Bright field pictures of melanoma cells upon SB9 treatment. (p) Growth rates of melanoma cells upon SB9 treatment. (n=3 independent experiments). (q,r) Migration and pro-inflammatory gene signature of fibroblasts treated with SB9. Data were normalized to GAPDH (n= independent experiments). In all relevant panels error bars represent ± SEM; indicates p<.5. WWW.NATURE.COM/NATURECELLBIOLOGY
a MAPK signaling secreted Secreted mirnas 76 exosomes WNT signaling b whole cell mature early Melanoma related mirnas 3 let-7a mir-5b mir- mir-9a 5 let-7b mir-7 mir-6a mir-3a mir-6 Relative mir expression... c f Relative mature mir expression in fibroblasts 8 6 d Fold Change (/ whole cell) 3.7 68.67 9.93 38.8 8.8 microrna name hsa-mir-3c hsa-mir-3 hsa-mir-98 hsa-mir-65-5p hsa-mir-63 e Relative mmu- expression in human fibroblasts 9 8 7 6 5 3 B6 Relative mature mir expression 8 6 primer hsa- primer mmu- Transfection: sc mimic hsa- mimic-mmu- g Relative mirna expression 8 6 normal melanoma mir- mir-let7b mir-9 mir-3c h Relative mirna expression 8 6 8 6 pre-mature mature secreted mir- mir-let7b mir-9a mir-3c i Relative mirna expression.6..8.. secreted secreted + Supplementary Figure 5 Identification of melanosomal mirnas and their trafficking into fibroblasts. (a) Venn diagram showing the overlap between the most highly expressed mirnas in, exosomes and melanoma-related mirnas (literature based,). (b) Yield of mature mirnas in preparations from whole cells, mature, and early. Data were normalized to levels of RNU6B. Error bars represent ± SEM (n=3 independent experiments). (c) Levels of indicated mature mirnas were evaluated in fibroblasts treated with or PBS () using RT-PCR. Data were normalized to RNU6B levels. Error bars represent ± SEM, indicates p<.5 (n= independent experiments). (d) Table of the five micrornas that are most up-regulated in mature compared to their levels in whole cells. (e) Expression level of mature mmu-mir- in human fibroblasts after treatment with B6F was determined using qrt-pcr. Data were normalized to RNU6B (n= independent experiments). (f) Human primary fibroblasts were transfected with human or mouse mir- mimic (hsa and mmu, respectively). qrt- PCR analysis of both mirnas. Data were normalized to RNU6B (n= independent experiments). (g) Mature mirnas levels in normal and melanoma (n= independent experiments). (h) Levels of mature mirnas in indicated fractions (n= independent experiments). (i) Levels of mature mirnas in secreted treated with the (SB9), normalized to RNU6B (n= independent experiments). WWW.NATURE.COM/NATURECELLBIOLOGY 5
a Relative proliferation 3..9..9..9 mir-3c 3 days b c d mir-3c h 6h h 8h 5µm mir-3c Relative area.5 mir-3c Relative mrna expression 5 3 mir-3c IL-β IL-6 IL-8 CXCLCXCL COX e normal melanoma DAPI mir- DAPI mir- HMB-5 5mm 5mm 5mm 5mm 5mm f Ret melanoma cell line Relative mature mir expression in human fibroblasts 3 mmu- g Relative mrna expression 8 6 normal fibroblasts CAFs melanoma α-sma cola collα..3.. F/8 CD5 Tramp h normal fibroblasts DAPI 5µm Tyrosinase 5µm DAPI Tyrosinase merged µm µm µm CAFs 5µm 5µm µm µm µm X X Supplementary Figure 6 Fibroblast reprogramming into CAFs is melanosomal mir- dependent. Fibroblasts treated with mir-3c were analyzed in comparison to fibroblasts treated with scrambled mirs. The fibroblasts were subjected to the following analyses: (a) growth rate (error bars represent ± SEM, n= independent experiments); (b) migration tested by scratch assay; (c) fibroblast collagen remodeling capacity examined by three-dimensional collagen contraction assay (error bars represent ± SEM, n=5 independent experiments); and (d) pro-inflammatory gene expression levels tested by qrt-pcr normalized to GAPDH levels (error bars represent ± SEM, indicates p<.5, n=3 independent experiments). (e) In situ hybridization analysis (green, x magnification) of mir- expression levels in healthy skin sample (left) and sections from the skin of a melanoma patient (right). Immunofluorescence staining of HMB-5 of the same magnification and location (red). DAPI-stained nuclei appear blue. (f) Mature mouse mir- levels (mmu-mir-) were measured in human fibroblasts that were treated with medium of Ret melanoma cell line or with medium. Data were normalized to RNU6B levels. Error bars represent ± SEM, indicates p<.5 (n=3 independent experiments). (g) PDGFRα-positive fibroblasts from local tumor stroma and melanoma cells from tumor were assessed for the expression of fibroblast markers (α-sma, collagen), macrophage markers (CD5, F/8), and melanoma markers (trpm, tyrosinase). Results were compared to the expression in normal fibroblasts sorted from mouse ears and normalized to GAPDH. Error bars represent ± SEM, indicates p<.5 (n= mice for treatment and for in 3 independent experiments). (h) Sorted fibroblasts from normal and stromal tissue stained for tyrosinase (green). DAPI-stained nuclei appear blue. Left panel: x magnification, right panel: x magnification. 6 WWW.NATURE.COM/NATURECELLBIOLOGY
a mir- - - b Fibroblasts upon melanosome treatment: IGF Fibroblasts upon mir- treatment: IGF ERK ERK c upregulation downregulation cell proliferation S cell proliferation IGFR HMB-5 DAPI S T S T S T E D S T 5µm 5µm 5µm 5µm d normal skin DAPI e E melanoma E T T T S 5µm D S D S HMB-5 T S IGFR µm normal IGFR µm melanoma mir- µm normal mir- melanoma 5µm 5µm IGFR f IGFR IGFR T S 5µm 5mm E D T S 5mm g invading seeded % of invading cells / seeded cells k 9 8 7 6 5 3 Melanoma-stroma communication by mirna trafficking s the primary tumor niche formation MNT- WM33 Epidermis Dermis h Relative MITF expression mir- DAPI 9 8 7 6 5 3 MNT WM33 E mir- DAPI D µm T S 5mm 5mm Normal Melanoma i Melanosomal mirnas WM33 7 3 Melanoma mir-5b related mir-7 mirnas let-7a In-situ Invasive Normal / Nevus melanoma melanoma MNT- 5 mir- Melanocyte Melanosome Fibroblast Cancer Associate Fibroblast Vascular endothelial cells Non invasive melanoma Invasive melanoma mir- IGFR j mirnas in correlation to MITF mir-.5 let7a mir-7 -.5 mir-5b - IGFR P-ERK CAFs Supplementary Figure 7 Melanosomal mir- induces CAF formation via up-regulation of MAPK signaling. (a) Hierarchical clustering analysis of mrna expression profiles of fibroblasts upon indicated treatments. Each row represents the mean of signal log ratios using a color-code scale. (b) Canonical pathway for IGFR signaling in fibroblasts upon indicated treatments, generated with IPA. Gene expression levels are relative to fibroblasts. Up- and downregulated genes are labeled red or green, respectively; predicted activation is shown in orange, inhibition in blue. Lines represent known interactions. Orange arrows: activation; blue arrows: inhibition; yellow arrows: inconsistency between observation and prediction; grey arrows: lack of supporting data. (c,d) Immunostaining of HMB-5 and IGFR in human specimens. Dashed lines separate tumor (T) and stroma (S); epidermis (E) and dermis (D) are indicated. DAPI-stained nuclei appear blue.. (e) Left: Immunostaining of HMB- 5 and IGFR in human specimens. Right: In situ hybridization of mir-. Dashed lines separate tumor (T) and stroma (S); epidermis (E) and dermis (D) are indicated. DAPI-stained nuclei appear blue (63X magnification). (f) Top: Immunostaining of HMB-5 and IGFR in human specimens. Bottom: In situ hybridization of mir-. Dashed lines separate tumor (T) and stroma (S); epidermis (E) and dermis (D) are indicated. DAPI-stained nuclei appear blue (63X magnification). (g) MNT- and WM33 melanoma cells invasion abilities, measured with a Matrigel invasion assay. Graph plots percent of invading cells. Representative images of invading and seeded cells are shown; DAPI-stained nuclei appear in blue. Error bars represent ± SEM, indicates p<.5 (n=3 independent experiments). (h) MITF expression levels in MNT- and WM33 melanoma cells normalized to GAPDH. Error bars represent ± SEM (n=3 independent experiments). (i) Venn diagram showing the overlap between the most highly expressed mirnas in MNT- and WM33 mature and melanoma-related mirnas (literature based 3, ). (j) Pearson s correlation for the expression level of each mirna relative to MITF in melanoma cell lines. mir-, Let7a, mir-7 and mir-5b are highlighted (correlations of p <.5). (k) A summary model - The formation of the melanoma primary niche by mirna trafficking. Graphical model describing our findings which demonstrate that melanoma cells directly affect the formation of the dermal tumor niche by mirna trafficking prior to invading the dermis. WWW.NATURE.COM/NATURECELLBIOLOGY 7
Supplementary Figure b: Tyrosinase Chemiluminescence: Rab7a Chemiluminescence: C PM MM WC M WC C PM MM WC M WC C: Cytoplasm PM: Pre-mature MM: Mature WC: Whole cell M: Melanosomes C: Cytoplasm PM: Pre-mature MM: Mature WC: Whole cell M: Melanosomes Photo: Photo: 6 kda kda kda 7 kda 5 kda kda kda 5 kda 5 kda Supplementary Figure 8 Unprocessed scans of western blots 8 WWW.NATURE.COM/NATURECELLBIOLOGY
Supplementary Figure e: Tyrosinase Chemiluminescence: DCT Chemiluminescence: Mel Exo Mel Mel Exo Mel Mel: Melanosomes Exo: Exosomes Mel: Melanosomes Exo: Exosomes Photo: 6 kda kda kda 7 kda 5 kda kda kda 5 kda 5 kda Photo: 6 kda kda kda 7 kda 5 kda kda kda 5 kda 5 kda Supplementary Figure 8 continued WWW.NATURE.COM/NATURECELLBIOLOGY 9
Supplementary Figure e: GPNMB CD63 Chemiluminescence: Ex Mel Chemiluminescence: Mel Exo Mel Mel: Melanosomes Exo: Exosomes Mel: Melanosomes Exo: Exosomes Photo: Photo: 6 kda kda kda 7 kda 5 kda kda 6 kda 7 kda 5 kda kda kda 5 kda 5 kda kda 5 kda Supplementary Figure 8 continued WWW.NATURE.COM/NATURECELLBIOLOGY
Supplementary Figure e: α-enolase HSC7 Chemiluminescence: Mel Exo Mel Chemiluminescence: Exo Mel Exo Mel HSC7 HSC7 Myosin Va Mel: Melanosomes Exo: Exosomes Mel: Melanosomes Exo: Exosomes Photo: Photo: 6 kda 7 kda 5 kda kda kda 5 kda 5 kda 6 kda kda kda 7 kda 5 kda kda kda Supplementary Figure 8 continued WWW.NATURE.COM/NATURECELLBIOLOGY
Figure 6d con mel con mel p-igfr IGFR con mel con mel 5 75 8 75 5 Supplementary Figure 8 continued WWW.NATURE.COM/NATURECELLBIOLOGY
Figure 6d p-igfr sc sc IGFR sc sc 8 75 5 Supplementary Figure 8 continued WWW.NATURE.COM/NATURECELLBIOLOGY 3
Figure 6d Tgf b Tgf b p-erk SC SC SC SC Tgf b Tgf b SC SC SC SC ERK 75 5 Supplementary Figure 8 continued WWW.NATURE.COM/NATURECELLBIOLOGY
Figure 6d p-erk con mel mel con mel mel ERK Supplementary Figure 8 continued WWW.NATURE.COM/NATURECELLBIOLOGY 5
Supplementary Figure 6d: 5 mir- mir- mir- mir- MAPK p-erk 5 mir- mir- mir- MAPK mir- ERK 5 con mel mel con mel mel p-erk 5 con mel mel con mel mel ERK Supplementary Figure 8 continued 6 WWW.NATURE.COM/NATURECELLBIOLOGY
Supplementary Figure 6d 5 mir- mir- mir- mir- p-erk 5 mir- mir- mir- mir- ERK MAPK MAPK 5 mel mel mel mel MAPK p-erk 5 mel mel mel mel MAPK ERK Supplementary Figure 8 continued WWW.NATURE.COM/NATURECELLBIOLOGY 7
Supplementary Table Primary fibroblasts %of most differentially expressed mrna Data Sets Supplementary Table Ingenuity pathway analysis Data Sets Supplementary Table 3 mirna profiling Data Sets Supplementary Table Overlap of downregulated mrna upon mir- and Data Sets. Supplementary Table 5 mirna profiling secreted vesicles Data Sets Supplementary Table 6 mirna profiling various melanoma Data Sets Supplementary Table 7 Oligonucleotides Data Sets Supplementary Table 8 Statistics Source Data Supplementary Table 9 Antibodies list Supplementary Video Isolated liposomes 8 WWW.NATURE.COM/NATURECELLBIOLOGY