Fig. 1

a. Proliferative (PRO) and invasive (INV) subpopulations were identified from the ZMEL1 zebrafish melanoma cell line, which was originally isolated from a transgenic zebrafish and can be transplanted into transparent casper zebrafish. b-c. Tracking of individual cells by time-lapse microscopy (both p<0.001 by linear regression, N=4 independent experiments). b. Growth curves (mean ± SE of mean, smoothed with moving window average of 5 time points) and doubling time (mean [95% CI]: 27.0 h [26.9, 27.1] vs. 29.7 h [29.6, 29.9] for ZMEL1-PRO and -INV, respectively). c. (left) Representative displacements of 500 tracks, and (right) model estimates ± 95% CI for alpha, the slope of the log-log plot of mean squared displacement vs. lag time (tau) for each ZMEL1-PRO and -INV. Larger alpha indicates more persistent motion, with α=1 for diffusive and α=2 for projectile motion. d. (left) The INV signature (Hoek et al., 2006) was the top gene set by Gene Set Association Analysis (GSAA) of ZMEL1-INV vs. -PRO RNA-seq. (Right) Dual waterfall plot of GSAA ranked by false discovery rate (FDR). Literature PRO/INV gene sets are indicated with an asterisk and colored according to FDR. e. Heatmap of genes in Hoek INV signature that are differentially expressed between ZMEL1-PRO and -INV (log₂ fold change cutoff ± 1.5, p_adj < 0.05). Human ortholog gene names are used for clarity (see Figure S1e for zebrafish gene names). f. Segmentation of representative images of ZMEL1-PRO and -INV tumors and distant metastases (e.g. to caudal region [box]) at 3 days post-transplant (3dpt). Original images shown in Figure S1e. g. Quantification of caudal metastases seeded by ZMEL1 populations at 3 dpt (OR [95% CI]: 11.62 [1.43, 94.53]; p=0.022 by logistic regression; N=3 independent experiments with PRO/INV 10/10, 31/33, and 13/13 fish per group, respectively; n=110 fish total; plot shows mean ± SD). h. Representative composite images at 1 dpt from time lapse confocal microscopy of ZMEL1 cells transplanted intravenously in larval zebrafish. Arrowhead indicates group of cells invading from the notochord (NC) and caudal hematopoietic tissue (CHT) into the tail fin mesenchyme (TF). Images are representative of n=13 fish per cell type. See Video S1 for full time lapse. i. Quantification of caudal tissue invasion by imaging at 4-6dpt (N=3 independent experiments with PRO/INV 23/23, 21/21, and 19/23 fish per group, respectively; OR [95% CI]: 13.58 [5.56, 33.18]; p<0.001 by logistic regression, plot shows mean ± SD). j. Relative number of ZMEL1-PRO and -INV cells isolated and quantified by flow cytometry from primary tumors and metastases of fish transplanted with a 1:1 mixture of ZMEL1-PRO and -INV (primary tumors from n=6 fish; metastases from n=4 fish; p= 0.51 and p=0.031, respectively, by one-sample two-sided t-test with Bonferroni correction). See also Figure S1, Tables S1, S5, S6, and Video S1.