Fig. 3.

Characteristics of CD24⁺cells isolated from osteosarcoma cells. (a)The mRNA expression of OCT4, SOX2, NANOG, and BMI1 in CD24⁺ cells sorted by FACS from MG-63 and MNNG/HOS cells was higher than in CD24⁻cells (***p < 0.001; **p < 0.01; and *p < 0.05). Error bars represent the standard deviation (SD) from at least three independent experiments. (b) The proliferative rate of CD24⁺ cells isolated from MG-63 and MNNG/HOS cells was higher than CD24⁻cells. Independent experiments were repeated three times. Data are presented as the mean ± standard deviation (***p < 0.001; **p < 0.01; and *p < 0.05). (c and d) Compared with CD24⁻ osteosarcoma cells, CD24⁺ cells exhibited stronger (c) invasive and (d) migratory capacities. Ten high power fields were selected for comparison. Data are presented as the mean ± standard deviation (***p < 0.001; **p < 0.01; and *p < 0.05). (e and f) (e) CD24⁺ osteosarcoma cells from MNNG/HOS cells showed a stronger tumorigenic capacity than CD24⁻ osteosarcoma cells. Representative images of CD24⁺and CD24⁻ cell-induced tumour formation at 8 weeks after 1 × 10⁴ cell injection. a: The whole image of the three-dimensional CT reconstruction. b: Cross section. c: Coronal section. d: Sagittal section. White arrow showing cortical destruction. (f) Corresponding paraffin-embedded tissues were processed for H&E staining (scale bar = 50 µm; magnified: scale bar = 20 µm) (see Table S5 for relevant details). (g and h) (g) Representative images of CD24⁺and CD24⁻ MNNG/HOS cell-induced lung metastasis at 8 weeks after 1 × 10⁴ cell injection. a: In vivo bioluminescence imaging of the mice. b: Lung tissues isolated from the mice (arrows indicate the pulmonary metastases). c: In vitro bioluminescence imaging of isolated lung tissues. (h) H&E staining of the corresponding paraffin-embedded lung tissues from mice (scale bar = 200 µm; magnified: scale bar = 50 µm) (see Table S7 for relevant details).