Fig 2: EMT genes regulated by ROCK1/2 control amoeboid invasive features.a Heatmap displaying enrichment scores for differentially expressed EMT and metastasis-related signatures in amoeboid A375M2 cells compared to A375P cells or to A375M2 cells treated with ROCKi (H1152 and Y27632) or blebbistatin using ssGSEA. b Representative phase-contrast images of A375M2 cells on top of collagen I matrix (left) and immunoblots of p-MLC2 in A375M2 and WM1361 cells (right) after 4 h treatment with ROCKi (H1152) (n = 3). Scale bar, 50 μm. c Fold regulation of EMT-related gene expression from EMT-directed qPCR array in A375M2 and WM1361 cells treated with ROCKi (H1152) for 4 h (n = 4). d Representative confocal images of p-MLC2 and F-actin staining in WM1361 cells on collagen I matrix after WNT11 knockdown (n = 3). Scale bar, 20 μm. e Quantification of cell morphology (>280 cells pooled from n = 3) and f p-MLC2 immunofluorescence signal normalized by cell area (>85 cells pooled from n = 3) in WM1361 cells on collagen I matrix after depletion of indicated genes. g Representative confocal images (left) and quantification (right) of adhesion of WM1361 cells to a monolayer of keratinocytes after depletion of indicated genes (n = 5 for SERPINE1, n = 4 for TCF4, n = 3 for WNT11, WNT5B, AHNAK and CAV2). Scale bar, 20 μm. h Representative confocal images (left) and quantification (right) of 3D invasion index through a collagen I matrix of WM1361 cells after depletion of indicated genes (n = 3). Scale bar, 50 μm. i Summary table of amoeboid functional assays on WM1361 cells after SERPINE1, WNT11, WNT5B, AHNAK, TCF4 and CAV2 knockdown. “+” sign indicates a significant phenotype of the corresponding gene knockdown. c, g, h Graphs show mean ± s.e.m. e, f Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show minimum and maximum range of values. b–h n means number of independent biological experiments. c Two-tailed t-test with Benjamini, Krieger and Yekutieli correction for multiple comparisons. e, f Kruskal–Wallis test with Benjamini, Krieger and Yekutieli correction. g, h One-way ANOVA with Benjamini, Krieger and Yekutieli correction. For all graphs, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The exact significant p values for *p, **p and ***p are provided in Supplementary Table 1.

Fig 3: FZD7 downstream of WNT11 supports melanosphere formation and amoeboid invasion via DAAM1.a, b Heatmap representing log2 fold change in expression of non-canonical Wnt receptors in a A375M2 cells compared to A375P cells or A375M2 cells treated with ROCKi (H1152 and Y27632) and blebbistatin and in b metastatic versus primary melanoma samples from indicated studies from GEO and TCGA databases. c–f After FZD7 or RYK knockdown in A375M2 and WM1361 cells, quantification of c sphere formation index (n = 4 for A375M2, n = 3 for WM1361), d cell viability (n = 5 for A375M2, n = 3 for WM1361), e cell morphology (>300 cells pooled from n = 3) and f p-MLC2 levels (n = 3 for A375M2, n = 4 for WM1361). g Quantification of 3D invasion index through a collagen I matrix after FZD7 knockdown in A375M2 (n = 5) and WM1361 cells (n = 3). h–j After PLCB1 or DAAM1 knockdown in A375M2 and WM1361 cells, quantification of h cell morphology (>250 cells pooled from n = 3), i sphere formation index (n = 4) and j cell viability (n = 6 for A375M2, n = 3 for WM1361). k Quantification of 3D invasion index through a collagen I matrix after DAAM1 knockdown in A375M2 (n = 4) and WM1361 cells (n = 3). l Representative phase-contrast images (left) and quantification of cell morphology (right) of A375P cells on collagen I matrix after WNT11 stimulation and PLCB1 or DAAM1 knockdown (>400 cells pooled from n = 5). Scale bar, 100 μm. m, n Representative immunoblots (top) and quantification (bottom) of m RhoA-GTP in pulldown samples and total RhoA in total lysate (n = 3) and n p-MLC2 levels (n = 4) in A375P cells after WNT11 stimulation and DAAM1 knockdown. c, d, f, g, i–k, m, n Graphs show mean ± s.e.m. e, h, l Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show minimum and maximum range of values. c–n n means number of independent biological experiments. a, g, k Two-tailed t-test. b Two-tailed t-test with Welch’s correction. c, d, f, i, j One-way ANOVA with Dunnett post-hoc test. e, h Kruskal–Wallis with Dunn’s multiple comparison test. l Kruskal–Wallis test with Benjamini, Krieger and Yekutieli correction. m, n One-way ANOVA with Tukey post-hoc test. For all graphs, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The exact significant p values for *p, **p and ***p are provided in Supplementary Table 1.

Fig 4: Analysis of the invasive front of human primary melanomas.a–i Representative images (left) and quantification (right) of a melanoma cell shape score, b H-score of p-MLC2 staining, c ki-67 positive cells, H-score of d DAAM1, e WNT11, f WNT5B, g ALDH1A1, h CD44 and i NANOG staining in matched TB and IF from primary melanomas. j Principal component analysis (PCA) based on the expression of amoeboid (cell shape and p-MLC2), proliferative (ki-67), non-canonical Wnt pathway (WNT11, WNT5B and DAAM1) and cancer stem cell-related (ALDH1A1, CD44 and NANOG) markers assessed by immunohistochemistry in matched TB and IF from primary melanomas. Percentage of variation explained by each component is given in the axis labels. k, l Kaplan–Meier survival curves of k overall survival and l disease-free survival according to ALDH1A1 protein expression in the IF from our cohort of primary melanomas. ALDH1A1 expression was categorized as low or high using the median expression. a–l 53 primary melanomas. a, e–i Scale bar, 100 μm; inset, 50 μm. b–d Scale bar, 300 μm. a–i Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show the minimum and maximum range of values. a, b, d–f Two-tailed paired t-test. c, g–i Two-tailed Wilcoxon test. j Scatter plot showing principal components 1 (PC1) and 2 (PC2). k, l Log-rank test. Human schematic in this figure was created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).

Fig 5: Analysis of the invasive front of human metastatic melanomas.a–i Representative images (left) and quantification (right) of a melanoma cell shape score, b H-score of p-MLC2 staining, c ki-67 positive cells, H-score of d DAAM1, e WNT11, f WNT5B, g ALDH1A1, h CD44 and i NANOG staining in matched TB and IF from melanoma metastases. j Model summarizing the findings of this study. The IF of human primary melanomas is enriched in cells with rounded-amoeboid morphology, high levels of Myosin II, proliferative, non-canonical Wnt and cancer stem cell-related markers. This phenotype is recapitulated and further enriched in melanoma metastasis. WNT11/5B activate FZD7 and DAAM1 to control Rho activity and then ROCK1/2-Myosin II levels. All these signalling components have an impact on amoeboid features and tumour initiation in melanoma both in vitro and in vivo. Specifically, DAAM1 plays an essential role in tumour and metastasis initiation and subsequent metastatic outgrowth by sustaining the amoeboid phenotype. a–i 45 metastatic melanomas. a, e–i Scale bar, 100 μm; inset, 50 μm. b–d Scale bar, 300 μm. a–i Box limits show 25th and 75th percentiles, the horizontal line shows the median, and whiskers show minimum and maximum range of values. a, b, d–f Two-tailed paired t-test. c, g–i Two-tailed Wilcoxon test. Human schematic in this figure was created using Servier Medical Art templates licensed under a Creative Commons Attribution 3.0 Unported License (https://smart.servier.com).