Fig. 3. SOX9^high tumor cells have increased metastasis-initiating capacity.

(A) Correlation between SOX9 levels and functional states in breast tumors at the single-cell level as determined by CancerSEA. EMT, Epithelial-Mesenchymal Transition. (B) Transwell migration assay of C3/TAg primary tumor cells (n = 5 tumors). (C) Transwell invasion assay of C3/TAg primary tumor cells (n = 5 tumors). (D) Representative images and frequencies of organoids with the invasive morphology in collagen gel (n = 3 tumors). (E) Lung metastasis-initiating ability of Sox9^low and Sox9^high C3/TAg primary tumor cells by tail vein injection. Each line indicates one experiment with a distinct tumor (n = 4). (F) Lung metastasis-initiating ability of Sox9^neg, Sox9^low, and Sox9^high PyMT tumor cells by tail vein injection. Metastatic nodules were determined after 14 weeks (triangles and circles representing two different donors). (G) Lung metastasis-initiating ability of Sox9^neg, Sox9^low, and Sox9^high PyMT tumor cells by tail vein injection. Metastatic nodules were determined 14 weeks after injection (n = 3). All data are represented as means ± SEM. P values were determined by two-tailed t test [(B), (C), (D), (E), and (G)] or one-way ANOVA with Tukey’s test (F). ***P < 0.001, **P < 0.01, and *P < 0.05.