a, Schematic representation of the generation of genetically engineered primary melanomas in adult zebrafish (Methods). b, Proportion of nectin1a and nectin1b mutant alleles in 65 primary zebrafish melanomas expressing a CRISPR vector targeting nectin1a and nectin1b. Bar represents median; paired two-tailed t-test. c, Box-and-whisker plot representing the number of nectin1a and nectin1b mutant alleles per primary zebrafish melanoma described in panel b (min-max, 1st and 4th quartiles, median, n = 65; paired two-tailed t-test). d, Type and proportion of nectin1a and nectin1b mutant alleles in 10 representative primary zebrafish melanomas. D, deletion, I, insertion; numbers denote affected base pairs. The last section in each bar represents all other minor alleles. e, Tumor-free survival curves of Tg(mitf:BRAFV600E);tp53−/− zebrafish injected with vectors targeting either nectin1 or a control gene (pooled data of three independent experiments; log-rank test). f, Representative images of sections of control- or nectin1-knockout (KO) primary zebrafish melanomas, stained for phospho-histone3 (p-H3) by immunohistochemistry (IHC). Scale bar, 20 μm. g, Quantification of overall p-H3 staining intensity in 8 nectin1-wildype and 10 nectin1-knockout primary zebrafish melanomas (mean ± s.d.; two-tailed t-test). NS, not significant. h, Pictures of organs presenting with disseminated tumor cells (arrowheads) in hematoxylin/eosin-stained sections of 16-week-old zebrafish bearing nectin1-knockout melanoma. Scale bar, 20 μm. i, Number of 16-week-old zebrafish with disseminated melanoma cells in the indicated organs. j, Quantification of organ involvement in 16-week-old zebrafish bearing control (n = 11) or nectin1-knockout (n = 10) melanoma (mean ± s.d.). Two-tailed t-test. k, Schematic representation of the melanoma spreading assay in adult zebrafish (top) and representative images of casper zebrafish 7 or 21 days after transplantation with control- or nectin1-knockout melanoma cells (bottom). Insets show ×2 magnification views. Arrows indicate patches of disseminated melanoma cells. Scale bar, 1 cm. l, Quantification of the proportion of secondary recipients of control- (n = 10) or nectin1-knockout (n = 9) tumors showing tumor spreading (mean ± s.d.; two-tailed t-test). m, Cell count of control- and nectin1-knockout primary zebrafish melanoma lines in the presence or absence of zebrafish (zf) culture media. Data are presented as mean ± s.d. of three independent experiments.
|
