Fig. 4: The progeny of hypoxia-induced polyploid cells preferentially metastasize to bone.

a Design of the experiments for investigating the metastatic properties of the progeny of diploid (2n) and teraploid (4n) cell fractions isolated from normoxic (NOR) or hypoxic (HYP) SK-ES-1 cells using the FUCCI Cell Cycle Sensor followed by DNA staining and FACS. b Violin plot shows the analysis of nuclear area of the isolated diploid and tetraploid populations from NOR or HYP cells at passages 1 or 6–10 after sorting (Control SK-ES-1 n = 137; passage 1: NOR-2n n = 384, NOR~4n n = 282, HYP-2n n = 334, HYP~4n n = 143; passage 6–10: NOR-2n n = 161, NOR~4n n = 132, HYP-2n n = 173, HYP~4n n = 162). The red lines represent the median; the black lines represent the quartiles. P < 0.0001 vs. SK-ES-1 cells; one-way ANOVA followed by Dunnett’s test. c Analysis of mitotic segregation errors in NOR-2n (n = 24) or HYP~4n SK-ES-1 cells (n = 38). Two-sided Fisher’s exact test. d Flow cytometry analysis of DNA content in the original cell line and the cells cultured from parental and HYP~4n SK-ES-1 xenografts. e Representative images of FISH with CEN4/Y5R probes in the original SK-ES-1 cells (n = 53) or cells cultured from HYP~4n bone metastasis (n = 168), followed by the analysis of CEN4 numerical aberrations. Two-sided Fisher’s exact test. Scale bar: 10 μm. f Analysis of metastasis development in mice with control (n = 10) or HYP~4n (n = 7) SK-ES-1 xenografts; Generalized estimating equation (GEE) test. g, h Analysis of number of metastases (g) and the percentage of animals with bone metastases (h) in mice with SK-ES-1 xenografts developed from control (n = 10) or sorted cell fractions (NOR-2n n = 4, NOR~4n n = 3, HYP~4n n = 7). Error bars indicate standard error of the mean. Two-tailed Mann–Whitney test (g); One-sided χ² test (h). i Representative image of multiple HYP~4n osseous metastases in the hind limb of mice bearing HYP~4n SK-ES-1 xenografts (n = 5). Scale bar: 2000 μm. T - tumor.