Fig 2: Role of IGF1 signaling in zebrafish and human melanoma.(a) Tumor-free survival curves of Tg(mitf:BRAFV600E);tp53−/− zebrafish injected with a vector targeting nectin1 and vectors expressing either GFP or CD8-IGF1R (IGF1R*). Pooled data of 2 independent experiments. Log-rank test. (b) Quantification of overall p-H3 staining intensity in 8 nectin1-knockout/GFP and 8 nectin1-knockout/IGF1R* primary zebrafish melanomas. Mean ± SD. Two-tailed t-test. (c) Quantification of organ involvement in 16-week-old zebrafish bearing nectin1-knockout/GFP (n = 8) or nectin1-knockout/IGF1R* (n = 8) primary melanoma. Two-tailed t-test. (d) Progression-free survival curves of patients with melanoma stratified according to IGF1R mRNA expression (top and bottom n = 104 samples). TCGA cohort. Log-rank test. (e) Immunofluorescence analysis of α-catenin (green) and phospho-IGF1R (red) on a tissue section of human melanoma. Scale bar: 10 μm. Dotted line demarcates areas of high and low IGF1 signaling. Arrows point to adherens junctions. Representative example of 9 independent tumors. (f) Histogram representing signal intensities of α-catenin (green) and phospho-IGF1R (red) along the dotted line in the human melanoma tissue section shown on the left. Note the mutual exclusivity of the two signals. Source data

Fig 3: Implication of IGF1 in the regulation of adherens junctions between melanoma cells.(a) Immunofluorescence analysis of a-catenin (green) in A375 human melanoma cells stably expressing a control shRNA (shCTRL) or an shRNA against NECTIN1 (shNECTIN1) and cultured in the presence or absence of serum (FBS), or in the presence of dextran-treated/charcoal-stripped serum (treated FBS) for 12 hours. Scale bar: 10 µm. Representative of 4 independent experiments. (b) Western Blot analysis of FAK and SRC phosphorylation levels in the cells described in a cultured in the presence or absence of serum (+FBS or -FBS), or in the presence of dextran-treated/charcoal-stripped serum (tFBS) for 12 hours. Representative example of 3 independent experiments. (c) Most represented categories of biological processes in a Gene Ontology (GO) analysis of the transcriptional response to serum starvation in A375 human melanoma cells. The right column indicates the number of significant GO terms assigned to each category. (d) Enrichment plot of an IGF1/IGF2 signature found among the most significantly enriched pathways in A375 cells cultured in the presence vs absence of serum by GSEA. The FDR p-value was calculated by GSEA using a permutation test. (e) List of secreted factors tested for their ability to prevent the formation of adherens junctions between melanoma cells upon serum withdrawal. (f) Immunofluorescence analysis of a-catenin (green) and F-actin (red) in A375 cells cultured in the presence or absence of serum (FBS), complemented or not with various growth factors, for 12 hours. Scale bar: 10 µm. Representative of 3 independent experiments. (g) Western Blot analysis of IGF1R in A375 cells stably transduced with a doxycycline-inducible vector expressing either GFP or a constitutively active form of IGF1R (CD8-IGF1R, IGF1R*) and treated with doxycycline for 12 hours. CD8-IGF1R (estimated size: 70 kDa) appears below endogenous IGF1R (estimated size: 95 kDa). Representative example of 3 independent experiments. Source data

Fig 4: A decrease in IGF1 signaling triggers the formation of adherens junction between NECTIN1-wild-type cells or the migration and dissemination of NECTIN1-deficient cells.a, Immunofluorescence analysis of a-catenin (green) and F-actin (red) in A375 human melanoma cells in the presence or absence of serum (FBS) (blue, DAPI), complemented or not with 100 ng ml-1 IGF1 for 12 h. Scale bar, 10 µm. Data are representative of four independent experiments. b, Immunofluorescence analysis of a-catenin (green) and F-actin (red) in A375 human melanoma cells expressing or not a constitutively active form of the IGF1 receptor (CD8-IGF1R, IGF1R*), and cultured in the absence of serum (FBS) for 12 h (blue, DAPI). Scale bar, 10 µm. Data are representative of three independent experiments. c, Immunofluorescence analysis of a-catenin (green) and F-actin (red) in A375 human melanoma cells cultured in the presence of serum (FBS) and treated with DMSO (vehicle) or with two different IGF1R inhibitors (IGF1Ri#1: Linsitinib, 1 µM; IGF1Ri#2: GSK1838705A, 1 µM) for 12 h (blue, DAPI). Scale bar, 10 µm. Data are representative of four independent experiments. d, Migration of six human melanoma cell lines conditionally expressing GFP or a constitutively active form of IGF1R (CD8-IGF1R, IGF1R*), and stably expressing an shRNA directed against NECTIN1 (shNECTIN1) relative to cells expressing a control shRNA (shCTRL) in a transwell assay. Cells were allowed to migrate for different times depending on the cell line (Extended Data Fig. 4i and Methods). Data represent mean ± s.d. of four independent experiments (paired two-tailed t-test). e, Migration of A375 human melanoma cells stably expressing either a control shRNA (shCTRL) or an shRNA directed against NECTIN1 (shNECTIN1) in a transwell assay after 12 h of serum starvation in the presence or absence of 100 ng ml-1 IGF1. Cells were allowed to migrate for 6 h. Data represent mean ± s.d. of three independent experiments (paired two-tailed t-test). f, Representative images of adult casper zebrafish transplanted with nectin1-knockout primary zebrafish melanoma cells expressing GFP or a constitutively active form of IGF1R (CD8-IGF1R, IGF1R*), 21 days after injection. Insets show ×2 magnification views. Arrowhead indicates patches of disseminated melanoma cells. Scale bar, 1 cm. g, Quantification of the proportion of secondary recipients of nectin1-knockout tumors expressing GFP (n = 6) or nectin1-knockout tumors expressing CD8-IGF1R (IGF1R*) (n = 5) showing tumor spreading. Data represent mean ± s.d. (two-tailed t-test).Source data