Targeting of bone-derived insulin-like growth factor-II by a human neutralizing antibody suppresses the growth of prostate cancer cells in a human bone environment

Abstract

Purpose: Advanced prostate cancer frequently involves the bone, where the insulin-like growth factor (IGF)-II is abundant. However, the importance of IGF-II in bone metastasis from prostate cancer is uncertain. The present study was aimed at examining the therapeutic importance of targeting IGF-II in bone metastases from prostate cancer.

Experimental design: We investigated whether inhibiting IGF-II using a human neutralizing antibody (m610) suppresses the growth of prostate cancer cells in a human bone environment. Human MDA PCa 2b prostate cancer cells were inoculated into human adult bone implanted into mammary fat pad of nonobese diabetic/severe combined immunodeficient mice or inoculated into mammary fat pad of the mice without human bone implantation. The mice were treated with m610 or a control antibody (m102.4) once weekly for 4 weeks immediately after inoculation with MDA PCa 2b cells.

Results: Histomorphologic examination indicated that m610 treatment significantly decreased the MDA PCa 2b tumor area in the human bone compared with the control. Ki-67 immunostaining revealed that the percentage of proliferating cancer cells in the m610-treated bone tumor sections was significantly lower than that in the control. m610 had no effect on MDA PCa 2b tumor growth in the absence of implanted human bone. m610 prevented the in vitro IGF-II-induced proliferation of MDA PCa 2b cells.

Conclusions: Our results indicate that IGF-II plays an important role in the prostate cancer cell growth in human bone, suggesting that targeting it by neutralizing antibodies offers a new therapeutic strategy for bone metastasis from prostate cancer.

Fig. 1. Summary of treatment protocol

On day 0, NOD/SCID mice were either implanted with HAB into MFP tissues or not. After 4 weeks, MDA PCa 2b cells were inoculated into the implanted HAB or into MFP tissues of the mice without implantation. Weekly administration of m610 was started immediately after the cell inoculation. At 4 weeks after the treatment, the mice were sacrificed for histological analysis and measurement of serum PSA.

Fig. 2. M610 suppresses the growth of bone tumors from MDA PCa 2b cells in HAB

A representatively shows macroscopic images of the PSA-stained HAB sections from control and m610-treated groups. Localized PSA-positive MDA PCa 2b tumor foci were observed. Scale bars, 2 mm. The sections were histomorphologically analyzed for B, total tumor area and C, percentage of tumor area to total HAB tissue area. D, serum PSA values in blood samples of the groups. Control; control antibody group, m610 (1); low-dose (1 mg/kg) group, m610 (10); high-dose (10 mg/kg) group. Data are the means for 8 mice in each group; bars, ± SE. *P<0.05; **P<0.01, compared with the control.

Fig. 2. M610 suppresses the growth of bone tumors from MDA PCa 2b cells in HAB

A representatively shows macroscopic images of the PSA-stained HAB sections from control and m610-treated groups. Localized PSA-positive MDA PCa 2b tumor foci were observed. Scale bars, 2 mm. The sections were histomorphologically analyzed for B, total tumor area and C, percentage of tumor area to total HAB tissue area. D, serum PSA values in blood samples of the groups. Control; control antibody group, m610 (1); low-dose (1 mg/kg) group, m610 (10); high-dose (10 mg/kg) group. Data are the means for 8 mice in each group; bars, ± SE. *P<0.05; **P<0.01, compared with the control.

Fig. 3. IGF-1R and IR are expressed in MDA PCa 2b tumors in HAB

A, IGF-1R immunostained images of HAB sections from control and high-dose group are representatively shown. B, IR immunostained images. Scale bars, 100 μm. The higher magnification insets show the tumor cells with membranous and cytoplasmic immunostaining for IGF-1R and IR, respectively (Scale bars, 50 μm).

Fig. 4. M610 suppresses the proliferative status of MDA PCa 2b tumors in HAB

A, Ki-67 immunostained images of HAB sections from control and high-dose group are representatively shown. B, cleaved caspase-3 immunostained images. Scale bars, 100 μm. The percentages of positively immunostained cancer cells to the total cells were calculated. C and D, percentages of Ki-67-positive and cleaved caspase-3-positive cancer cells, respectively. CC-3, cleaved caspase-3. Data are the means; bars, ± SE. ***P < 0.001, compared with the control.

Fig. 4. M610 suppresses the proliferative status of MDA PCa 2b tumors in HAB

A, Ki-67 immunostained images of HAB sections from control and high-dose group are representatively shown. B, cleaved caspase-3 immunostained images. Scale bars, 100 μm. The percentages of positively immunostained cancer cells to the total cells were calculated. C and D, percentages of Ki-67-positive and cleaved caspase-3-positive cancer cells, respectively. CC-3, cleaved caspase-3. Data are the means; bars, ± SE. ***P < 0.001, compared with the control.

Figure 5. M610 does not suppress the growth of tumors from MDA PCa 2b cells in MFP

Histological analyses in MFP model. A, total tumor area. The data are the means for 4 mice in the control group and 5 mice in the m610-treated group; bars, ± SE. B, percentages of Ki-67-positive cancer cells.

Fig. 6. M610 inhibits IGF-2-induced cell proliferation and phosphorylations of IGF-1R/IR and Akt in MDA PCa 2b cells

A, MDA PCa 2b cells were treated for 48 hours with serum-free medium alone, with various concentrations of IGF-2, with the control antibody plus 100 ng/mL of IGF-2, or with various concentrations of m610 in the presence or absence of 100 ng/mL of IGF-2. The cells were counted using trypan blue dye exclusion. Data are the means of triplicate determinations and are representative of three independent experiments; bars, ± SE. ^#P < 0.05, compared with serum-free medium. **P < 0.01, compared with control antibody group. B, The cells were treated with 100 ng/ml of IGF-1 or IGF-2. Tyrosine phosphorylation of immunoprecipitated IGF-1R or IR were examined by a western blot analysis. WB, western blot; IP, immunoprecipitation; p-Tyr, phosphorylated tyrosine. C, The cells were pre-incubated with the indicated concentrations of m610, and 100 ng/mL of IGF-2 was added. IGF-1R/IR and Akt phosphorylation levels and the total protein levels in the lysates were examined by western blot analysis. p-, phosphorylated; t-, total.

Fig. 6. M610 inhibits IGF-2-induced cell proliferation and phosphorylations of IGF-1R/IR and Akt in MDA PCa 2b cells

A, MDA PCa 2b cells were treated for 48 hours with serum-free medium alone, with various concentrations of IGF-2, with the control antibody plus 100 ng/mL of IGF-2, or with various concentrations of m610 in the presence or absence of 100 ng/mL of IGF-2. The cells were counted using trypan blue dye exclusion. Data are the means of triplicate determinations and are representative of three independent experiments; bars, ± SE. ^#P < 0.05, compared with serum-free medium. **P < 0.01, compared with control antibody group. B, The cells were treated with 100 ng/ml of IGF-1 or IGF-2. Tyrosine phosphorylation of immunoprecipitated IGF-1R or IR were examined by a western blot analysis. WB, western blot; IP, immunoprecipitation; p-Tyr, phosphorylated tyrosine. C, The cells were pre-incubated with the indicated concentrations of m610, and 100 ng/mL of IGF-2 was added. IGF-1R/IR and Akt phosphorylation levels and the total protein levels in the lysates were examined by western blot analysis. p-, phosphorylated; t-, total.

Fig. 6. M610 inhibits IGF-2-induced cell proliferation and phosphorylations of IGF-1R/IR and Akt in MDA PCa 2b cells

A, MDA PCa 2b cells were treated for 48 hours with serum-free medium alone, with various concentrations of IGF-2, with the control antibody plus 100 ng/mL of IGF-2, or with various concentrations of m610 in the presence or absence of 100 ng/mL of IGF-2. The cells were counted using trypan blue dye exclusion. Data are the means of triplicate determinations and are representative of three independent experiments; bars, ± SE. ^#P < 0.05, compared with serum-free medium. **P < 0.01, compared with control antibody group. B, The cells were treated with 100 ng/ml of IGF-1 or IGF-2. Tyrosine phosphorylation of immunoprecipitated IGF-1R or IR were examined by a western blot analysis. WB, western blot; IP, immunoprecipitation; p-Tyr, phosphorylated tyrosine. C, The cells were pre-incubated with the indicated concentrations of m610, and 100 ng/mL of IGF-2 was added. IGF-1R/IR and Akt phosphorylation levels and the total protein levels in the lysates were examined by western blot analysis. p-, phosphorylated; t-, total.