CD137 promotes bone metastasis of breast cancer by enhancing the migration and osteoclast differentiation of monocytes/macrophages

Abstract

Rationale: Bone is one of the most common metastatic sites of breast cancer. CD137 (4-1BB), a member of the tumor necrosis factor (TNF) receptor superfamily, is mainly expressed in activated leukocytes. Previous study demonstrates the effect of CD137-CD137L bidirectional signaling pathway on RANKL-mediated osteoclastogenesis. However, the role of CD137 in bone metastasis of breast cancer needs further study. Methods: Stable monocyte/macrophage cell lines with Cd137 overexpression and silencing were established. Western blot, real-time PCR, transwell and tartrate-resistant acid phosphatase staining were used to detect the regulatory effect of CD137 on migration and osteoclastogenesis of monocytes/macrophages in vitro. Spontaneous bone metastasis mouse model was established, bioluminescent images, immunohistochemistry and histology assay were performed to detect the function of CD137 in bone metastasis in vivo. Results: We found that CD137 promotes the migration of monocytes/macrophages to tumor microenvironment by upregulating the expression of Fra1. It also promoted the differentiation of monocytes/macrophages into osteoclasts at the same time, thus providing a favorable microenvironment for the colonization and growth of breast cancer cells in bone. Based on these findings, a novel F4/80-targeted liposomal nanoparticle encapsulating the anti-CD137 blocking antibody (NP-αCD137 Ab-F4/80) was synthesized. This nanoparticle could inhibit both bone and lung metastases of 4T1 breast cancer cells with high efficacy in vivo. In addition, it increased the therapeutic efficacy of Fra1 inhibitor on tumor metastasis. Conclusions: Taken together, these findings reveal the promotion effect of macrophage/monocyte CD137 on bone metastases and provide a promising therapeutic strategy for metastasis of breast cancer.

Keywords: Fra1; anti-CD137 antibody; bone metastasis; breast cancer; liposomal nanoparticles; metastatic niche..

Figure 1

Elevated serum sCD137L level in patients with metastatic breast cancer. A. Representative IHC staining of CD137 and CD137L in human breast cancer tissue array (left panel, scale bar: 50 μm) and the statistical analysis results for their expression pattern in breast tumor tissues (right panel). B. Statistical analysis results of OD450 value of serum sCD68 from normal female and female breast cancer (BC) patients measured by ELISA. Data are presented as mean ± SEM. C. Representative Western blot results of serum sCD137L in normal female and female breast cancer (BC) patients (left panel) and the statistical analysis results of Western blot, data are presented as mean ± SEM (right panel).

Figure 2

CD137 enhances the migration of monocytes/macrophages and promotes their differentiation into osteoclasts. A-B. Western blot results of CD137, Fra1, and renilla luciferase (Renilla luc) in RawRL cells. The mean value of relative fold change (RFC) for each blot is indicated at the bottom, n = 3. C-E. Schematic diagram to show the transwell assay of RawRL cells (C) and the statistical results for the change of migrated cell number (#) per image field from the transwell assay (D-E, n = 3). F. RNA-seq results for the differential expression genes associated with cell migration in RawRL-CD137 cells when compared with RawRL-Con. G. Statistical results for the change of migrated cell number per image field from the transwell assay of RawRL-CD137 cells that treated with αCD137 Ab, SKLB816 (Fra1 inhibitor) and the combination of αCD137 Ab and SKLB816, respectively. IgG treatment was used as the control, (n = 3). H-I. TRAP staining of RawRL cells which were treated with M-CSF (10 ng/mL) and RANKL (100 ng/mL) for 9 days. Left panel: Representative image of each group, the representative osteoclasts are indicated the red arrows, scale bar: 50 μm. Right panel: Statistical results for the number of TRAP positive osteoclasts per image field (n = 3). J. Real-time PCR assay of relative mRNA level change of osteoclast-related genes in RawRL cells after treatment with M-CSF (10 ng/mL) and RANKL (100 ng/mL) for 9 days (n = 3).

Figure 3

Monocyte/Macrophage expression of CD137 promotes bone metastases of breast cancer. A. Schematic diagram of experiment procedure. B. Representative IHC staining of Ki67 (upper panel, scale bar: 50 µm) and the statistical results for the percentage of Ki67 positive cells in 4T1FL-allografts of BALB/c mice (lower panel, n = 5 - 6 mice). C. BLI images of tumor-invasion lesions in each group (upper panel) at the end point of experiment and statistical results of normalized BLI signals (lower panel, n = 6 mice). D. Upper panel: Representative H&E staining image of bone lesions invaded by tumors in each group, scale bar: 200 μm. Low panel: Quantification of bone metastatic lesion number base on H&E staining, data are presented as mean ± SEM (n = 6 mice). E. Upper panel: TRAP staining of the osteolytic bone lesion from a representative mouse in each group, scale bar: 100 μm. Low panel: Statistical results of TRAP⁺ osteoclasts in osteolytic bone lesions (n = 7-11 bone lesions). F. Upper panel: Representative IHC staining of renilla luciferase⁺ cells in osteolytic metastasis areas, scale bar: 100 μm. Low panel: Statistical results of the percentage of renilla luciferase (RL)⁺ cells in osteolytic bone lesions (n = 6 mice). Abbreviation: d: day(s); w: week(s); ROI: region of interest; B: bone; T: tumor.

Figure 4

Increased targeting efficiency of NPs-αCD137 Ab PE-F4/80 in breast tumor tissues. A. Schematic diagram for the preparation of NPs-αCD137 Ab-F4/80. B. The encapsulation efficiency of NPs-αCD137 Ab-PE and NPs-αCD137 Ab-PE-F4/80. Data are presented as mean ± SEM (n = 3). C. Representative dynamic light scattering measurement for size distribution of NPs-αCD137 Ab-F4/80. D. The characterizations of the liposomal nanoparticles. Data are presented as mean ± SEM (n = 3). E. Schematic diagram of experiment procedure. F. Confocal images of anti-CD137 Ab-PE (upper panel, scale bar: 20 μm) and the statistical results for the relative fold changes of the concentration of anti-CD137 Ab-PE in 4T1FL-allografts of BALB/c mice that were treated with NPs-αCD137 Ab-PE or NPs-αCD137 Ab-PE-F4/80 for 6 hours (n = 4 mice, lower panel). G. Confocal images of anti-CD137 Ab-PE and CD68 (upper panel, scale bar: 20 μm) and the statistical results for the ratio of CD68⁺/DAPI⁺ and PE⁺CD68⁺/CD68⁺ cells in 4T1FL-allograft of BALB/c mice treated with NPs-αCD137 Ab-PE or NPs-αCD137 Ab-PE-F4/80 for 6 hours (lower panel, n = 4 mice). Abbreviation: d: day(s); i.v.: intravenous injection.

Figure 5

NPs-αCD137 Ab-F4/80 inhibit bone and lung metastases of breast cancer. A. Schematic diagram of experiment procedure. NPs-αCD137 Ab-F4/80 and their controls (PBS, NPs-IgG-F4/80, αCD137 Ab, NPs-αCD137 Ab) were administrated at the time points indicated by arrows. B. Left panel: BLI images of tumor-invasion lesions in each group at the end point of experiment. Right panel: Statistical results of normalized BLI signals (n = 5 mice). C. Left panel: Representative H&E staining image of bone lesions invaded by tumors from each group, scale bar: 200 μm. Right panel: Statistical results of bone metastatic lesion number, data are presented as mean ± SEM (n = 5 mice). D. Left panel: Representative TRAP staining of the osteolytic bone lesions from each group, scale bar: 100 μm. Right panel: Statistical results of TRAP⁺ osteoclasts in osteolytic bone lesions in each group (n = 2-5 bone lesions). E. Left panel: H&E staining of lung metastasis from a representative mouse in each group, scale bar: 200 μm. Right panel: Statistical results of lung metastatic foci number (data are presented as mean ± SEM) and lung metastatic area ratio in each group (n = 5 mice). Abbreviation: d: day(s); w: week(s); i.v.: intravenous injection.

Figure 6

Dual-target therapy against CD137 and Fra1 for breast cancer. A. Schematic diagram of experiment procedure. Combined therapy (NPs-αCD137 Ab-F4/80 plus SKLB816) and the controls (PBS, NPs-αCD137 Ab-F4/80, SKLB816) were administrated at time points indicated by the corresponding arrows. B. Left panel: BLI images of tumor-invasion lesions in each group at the end point of experiment. Right panel: Statistical results of normalized BLI signals (n = 4 mice). C. Left panel: H&E staining of lung metastasis from a representative mouse in each group, scale bar: 200 μm. Right panel: Statistical results of lung metastatic foci number (data are presented as mean ± SEM) and lung metastatic area ratio (n = 4 mice). D. Schematic summary of our proposed model: In tumor microenvironment, NPs-αCD137 Ab-F4/80 specifically block the CD137 signaling in the macrophages, which inhibit the differentiation of macrophages into osteoclasts, and decrease the expression of Fra1 to reduce the migration property of macrophages. Moreover, the NPs increase the anti-metastatic effect of SKLB816, which inhibits the expression of Fra1 in both cancer cells and macrophages. d: day(s); w: week(s); i.v.: intravenous injection.