Figure 8.
Figure 8. BMP7 suppresses tumor growth, but withdrawal induces tumor recurrence of CSCs in vivo. (A) CSCs isolated from PC3 mm were coinjected into tibiae of nude mice with control (scramble; n = 11) or BMP7–knocked down (shBMP7 #1, n = 14; #2, n = 10) HS5 cells. (left) BLI and x-ray radiography of bone lesions from representative mice in each group 4 wk after inoculation. Osteolytic lesions are indicated by arrowheads. (middle) Normalized BLI signals after 4 wk. *, P < 0.05 by Mann-Whitney test. (right) Representative images of immunohistochemical staining of tumors in the tibiae for p-p38 and NDRG1. (B) CSCs isolated from PC3 mm/shNDRG1 (n = 12) or PC3 mm/scramble (n = 12) were coinjected with HS5 into tibiae of nude mice. (left) BLI and x-ray radiography of representative mice in each group 4 wk after inoculation. Osteolytic lesions are indicated by arrowheads. (middle) BLI after 4 wk. **, P < 0.01 by Mann-Whitney test. (right) H&E staining of the tibiae from representative mice 4 wk after inoculation. The asterisk indicates a tumor. (C) Kaplan-Meier bone metastasis–free survival curve of mice after intracardiac injection with CSCs isolated from PC3 mm (left) or C4-2B (right) followed by treatment with vehicle or BMP7. Control, control vehicle treatment (PC3 mm, n = 9; C4-2B, n = 10); BMP7, continuous treatment with BMP7 (PC3 mm, n = 15; C4-2B, n = 10); BMP7 withdrawal, BMP7 treatment (2 wk for PC3 mm and 3 wk for C4-2B) followed by withdrawal of BMP7 (PC3 mm, n = 9; C4-2B, n = 10). ***, P = 0.0008; and **, P = 0.0012 versus control; and ###, P < 0.0001; and #, P = 0.0289 versus BMP7 by Log-rank test. (D) Effect of BMP7 on senescence was examined by SA–β-gal staining. The bone derivatives of PC3 mm (PC3 mm–BM) were cultured with (+/+) or without (−/−) BMP7 for 96 h. +/− cells were treated with BMP7 for 48 h followed by withdrawal of BMP7 and further incubation for 48 h (n = 3). ***, P < 0.001. (E, top) After injection of CSCs into mouse tibiae, BMP7 was administered daily i.v. Control, control vehicle treatment (n = 13); BMP7, continuous treatment with BMP7 (n = 10); withdrawal, 10-d treatment followed by withdrawal of BMP7 (n = 10). *, P < 0.05 versus control; ###, P < 0.001 versus BMP7 treatment. (bottom) Representative SA–β-gal staining (blue-green) of tumors in the tibiae of mice at the end point. Experiments in A–C and E were performed twice, the experiment in D was performed three times independently, and representative data are shown. Results are shown as mean ± SEM. Bars: (A, D, and E) 50 µm; (B) 200 µm.

BMP7 suppresses tumor growth, but withdrawal induces tumor recurrence of CSCs in vivo. (A) CSCs isolated from PC3 mm were coinjected into tibiae of nude mice with control (scramble; n = 11) or BMP7–knocked down (shBMP7 #1, n = 14; #2, n = 10) HS5 cells. (left) BLI and x-ray radiography of bone lesions from representative mice in each group 4 wk after inoculation. Osteolytic lesions are indicated by arrowheads. (middle) Normalized BLI signals after 4 wk. *, P < 0.05 by Mann-Whitney test. (right) Representative images of immunohistochemical staining of tumors in the tibiae for p-p38 and NDRG1. (B) CSCs isolated from PC3 mm/shNDRG1 (n = 12) or PC3 mm/scramble (n = 12) were coinjected with HS5 into tibiae of nude mice. (left) BLI and x-ray radiography of representative mice in each group 4 wk after inoculation. Osteolytic lesions are indicated by arrowheads. (middle) BLI after 4 wk. **, P < 0.01 by Mann-Whitney test. (right) H&E staining of the tibiae from representative mice 4 wk after inoculation. The asterisk indicates a tumor. (C) Kaplan-Meier bone metastasis–free survival curve of mice after intracardiac injection with CSCs isolated from PC3 mm (left) or C4-2B (right) followed by treatment with vehicle or BMP7. Control, control vehicle treatment (PC3 mm, n = 9; C4-2B, n = 10); BMP7, continuous treatment with BMP7 (PC3 mm, n = 15; C4-2B, n = 10); BMP7 withdrawal, BMP7 treatment (2 wk for PC3 mm and 3 wk for C4-2B) followed by withdrawal of BMP7 (PC3 mm, n = 9; C4-2B, n = 10). ***, P = 0.0008; and **, P = 0.0012 versus control; and ###, P < 0.0001; and #, P = 0.0289 versus BMP7 by Log-rank test. (D) Effect of BMP7 on senescence was examined by SA–β-gal staining. The bone derivatives of PC3 mm (PC3 mm–BM) were cultured with (+/+) or without (−/−) BMP7 for 96 h. +/− cells were treated with BMP7 for 48 h followed by withdrawal of BMP7 and further incubation for 48 h (n = 3). ***, P < 0.001. (E, top) After injection of CSCs into mouse tibiae, BMP7 was administered daily i.v. Control, control vehicle treatment (n = 13); BMP7, continuous treatment with BMP7 (n = 10); withdrawal, 10-d treatment followed by withdrawal of BMP7 (n = 10). *, P < 0.05 versus control; ###, P < 0.001 versus BMP7 treatment. (bottom) Representative SA–β-gal staining (blue-green) of tumors in the tibiae of mice at the end point. Experiments in A–C and E were performed twice, the experiment in D was performed three times independently, and representative data are shown. Results are shown as mean ± SEM. Bars: (A, D, and E) 50 µm; (B) 200 µm.

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