Extended Data Fig. 6: NETs promote tumour metastasis via CCDC25.
From: DNA of neutrophil extracellular traps promotes cancer metastasis via CCDC25
a, Primary breast cancer cells transduced with a control sgRNA or with one of two sgRNAs for CCDC25 were treated with 5 μg ml−1 NET-DNA or were untreated, and cell migration, adhesion and cytoskeleton remodelling were evaluated. n = 5 biologically independent experiments. Data are mean ± s.d. two-sided one-way ANOVA with Tukey test. b, NOD/SCID mice were intrasplenically injected with luciferase-primary breast cancer cells transduced with a control sgRNA or with one of two sgRNAs for CCDC25. Representative images (left) and quantification (right) of liver metastases after the indicated treatments are shown (n = 5 mice per group). Data are mean ± s.d., two-sided one-way ANOVA with Tukey test, **P = 0.0040 (sg1 versus ctrl-sg) and 0.0018 (sg2 versus ctrl-sg), ns = 0.8865. c, Representative images (left) and quantification (right) of lung metastases in wild-type (WT) PyMT mice and in PyMT;CCDC25−/− (PyMT-KO) mice pretreated with LPS or untreated (n = 5 mice per group). Data are mean ± s.d., two-sided one-way ANOVA with Tukey test, *P = 0.0244, ns >0.9999. d, Tumour burden in wild-type PyMT mice and PyMT;CCDC25−/− mice pretreated with LPS or untreated. n = 5 mice per group. Data are mean ± s.d., two-sided one-way ANOVA, ns = 0.9934. e, Representative images (left) and quantification (right) of liver metastases in C57BL/6 mice intrasplenically injected with tumour cells derived from the wild-type PyMT mice and PyMT-KO mice (n = 5 mice per group). Data are mean ± s.d., *P = 0.0215 determined by a two-tailed Student’s t-test. f, Validation of the polyclonal CCDC25 antibody. Indicated cell lysates from MDA-MB-231 cells transduced with a control sgRNA or with one of two sgRNAs for CCDC25 were subjected to western blot analysis probing with the polyclonal CCDC25 antibody. g. Representative bio-layer interferometry data of polyclonal antibody binding to recombinant protein CCDC25. The coloured lines show the data for five different concentrations of recombinant CCDC25 as indicated. h, Inhibitory effects of a polyclonal CCDC25 blocking antibody (5 μg ml−1) on NET-induced migration, adhesion and cytoskeleton arrangement of MDA-MB-231 cells. n = 5 biologically independent experiments. Data are mean ± s.d., two-sided one-way ANOVA with Tukey test. i, Representative images and quantification of liver metastases of NOD/SCID mice intrasplenically injected with luciferase-MDA-MB-231 cells, which were treated with IgG as a control or with a CCDC25 antibody. n = 6 mice per group. Data are mean ± s.d., **P = 0.0042 as calculated by a two-tailed Student’s t-test. Data in f, g are representative of three independent experiments.
