Myeloid cell reprogramming combined with zoledronic acid effectively suppresses bone metastasis

The bone metastasis microenvironment is predominantly composed of immunosuppressive myeloid cell populations. (A) t-SNE analysis of immune cell populations from bone marrow of tumor-free mice (tumor-free) and mice with established bone metastases (bone-mets), identifying distinct cell clusters (C01-C26). (B) t-SNE plots showing the distribution of immune cell clusters in the bone marrow from tumor-free mice and bone-mets mice. (C) Heatmap displaying the differential expression of selected marker across the identified immune cell clusters. (D, E) Quantification of the frequency of major immune cells in the bone marrow of tumor-free mice and bone-mets mice: (D) Monocytes/macrophages, neutrophils, and DCs; (E) B cells, CD4⁺ T cells, CD8⁺ T cells, NK cells, eosinophils, and basophils. (F) Schematic of the 4T1 bone metastasis model establishment and treatment (zoledronic acid, 100 μg/kg, i.p., 2 doses over 21 days). (G) Representative images and MRI scans of bone metastases at day 21. (H) Quantification of tumor burden. (I–N) Flow cytometry analysis of bone metastasis microenvironment for: (I) CD8⁺/CD3⁺ T cells; (J) NKp46⁺/CD45⁺ CD3⁻ NK cells; (K) MHC-II⁺/CD11c⁺ dendritic cells; (L) F4/80⁺/CD11b⁺ macrophages; (M) CD86⁺ and CD163⁺ macrophages; (N) Ly6G⁺/CD11b⁺ cells and CD101⁺ TANs. Bone-mets, bone-metastases; DC, dendritic cell; i.a., intraarterial; Mac, macrophage; Mono, monocyte; MRI, magnetic resonance imaging; NK, natural killer; TAM, tumor-associated macrophage; TAN, tumor-associated neutrophil; ZA, zoledronic acid. Significance is denoted as ns (not significant), *P < 0.05, and **P < 0.01.

DKK1 induces immature and immunosuppressive TANs in bone metastases. (A) Immature neutrophils sorted from mouse bone marrow were stimulated ex vivo with GM-CSF (50 ng/mL) or GM-CSF + DKK1 (50 ng/mL); mean fluorescence intensity (MFI) of Ly6G, CXCR2, CD101, and CXCR4 were assessed by flow cytometry. (B) Sorted immature neutrophils were treated with or without GM-CSF and DKK1 for 24 h, then co-cultured with primary CD8⁺ T cells (1:1) for 48 h. (C) The IFN-γ secretion was measured after co-culture. (D) CD25, CD69, and CD107a expression in CD8⁺ T cells were analyzed by flow cytometry. (E) After co-culture, CD8⁺ T cells were further co-cultured with CFSE-labeled tumor cells at a 5:1 ratio and tumor cell apoptosis was measured after 8 h. BM, bone marrow; DKK1, dickkopf-1; GM-CSF, granulocyte-macrophage colony-stimulating factor; MACS, magnetic activated cell sorting; Neu, neutrophil. Significance is denoted as ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Neutrophil-specific Ckap4 deletion promotes neutrophil maturation and restrains bone metastases tumor progression. (A) Schematic illustrating DKK1-CKAP4 interaction on neutrophils. (B, C) Ckap4^fl/fl and Ckap4^fl/flS100a8^cre mice were injected intra-arterially with LLC cells. Representative endpoint images and MRI scans of bone metastases are shown. (D) Quantification of tumor burden. (E) Proportion of mature neutrophils (CD11b⁺Ly6G⁺CD101⁺) in bone marrow analyzed by flow cytometry. (F–I) Flow cytometry analysis of bone TME for (F) CD86⁺/F4/80⁺, CD163⁺/F4/80⁺; (G) MHC-II⁺/CD11c⁺, CD103⁺ DCs; (H) CD8⁺/CD3⁺ T cells; (I) CD25⁺/CD8⁺, CD69⁺/CD8⁺, and CD107a⁺/CD8⁺ cells. BM, bone marrow; CKAP4, cytoskeleton-associated protein 4; DC, dendritic cell; DKK1, dickkopf-1; MRI, magnetic resonance imaging; TAM, tumor-associated macrophage; TAN, tumor-associated neutrophil. Data are presented as mean ± SEM. ns, not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.

DKK1 promotes the maturation and activation of osteoclast progenitors. (A) Osteoclast precursor cells from Balb/c mouse bone marrow were differentiated ex vivo with M-CSF (50 ng/mL) and RANKL (50 ng/mL), then treated with DKK1 (50 ng/mL) or DKK1 plus αDKK1 (10 μg/mL). (B, C) TRAP staining was used to quantify TRAP⁺ multinucleated cells with osteoclast-like features in each group. (D) Cytokines (IL-6, CCL2, and TNF-α) were measured after induction. (E) Gene set enrichment analysis (GSEA) of breast cancer GEO datasets (GSE14018, GSE14020, and GSE54323) showing the association between DKK1 expression and osteoclast- or osteoblast-related pathways. BM, bone marrow; DKK1, dickkopf-1; NES, normalized enrichment score; OCPs, osteoclast precursor cells; TRAP, tartrate-resistant acid phosphatase; αDKK1, DKK1 antibody. Significance is denoted as ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

DKK1 induces M2 polarization and immunosuppressive function of macrophages. (A) BMDMs from Balb/c mice were stimulated with LPS (50 ng/mL) with or without DKK1 (50 ng/mL), αDKK1 (10 μg/mL), and αCKAP4 (10 μg/mL). After 48 h, (B) the concentrations of IL-6, TNF-α, CCL2, and IL10 in culture supernatants were detected by CBA. The proportion of (C) CD80⁺ CD86⁺ and (D) CD163⁺ CD206⁺ macrophages was determined by flow cytometry. (E) BMDMs from C57BL/6 mice were stimulated with LPS (50 ng/mL) with or without DKK1 (50 ng/mL), αDKK1 (10 μg/mL), and αCKAP4 (10 μg/mL). After 48 h, (F) the concentrations of IL-6, TNF-α, CCL2, and IL10 in culture supernatants were detected by CBA. The proportion of (G) CD80⁺ CD86⁺ and (H) CD163⁺ CD206⁺ macrophages was determined by flow cytometry. (I) Integrative transcriptomic analysis of human breast cancer GEO datasets (GSE14018, GSE14020, and GSE54323) showed correlations between DKK1 expression and myeloid-derived chemokines. BM, bone marrow; DKK1, Dickkopf-1; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; NES, normalized enrichment score; αCKAP4, CKAP4 antibody; αDKK1, DKK1 antibody. Significance is denoted as ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Combining myeloid cell reprogramming with zoledronic acid suppresses tumor progression and remodels the bone metastasis immune microenvironment. (A, D) Schematic of syngeneic breast cancer bone metastasis model (A) or syngeneic lung cancer bone metastasis model (D) and treatment: αDKK1 [10 mg/kg i.p. (7 doses over 3 weeks)] and/or zoledronic acid [100 μg/kg (2 doses)]. (B, E) Representative images and MRI scans of 4T1 (B) or LLC bone metastases (E) at endpoint. (C, F) Quantification of 4T1 (C) or LLC tumor area (F) by MRI. (G-L) Flow cytometry analysis of 4T1 (G–I) and LLC bone metastasis microenvironment (J-L): (G, J) CD8⁺/CD3⁺, CD25⁺/CD8⁺, CD69⁺/CD8⁺, and CD107a⁺/CD8⁺ T cells; (H, K) Ly6G⁺/CD11b⁺ and CD101⁺/Ly6G⁺ neutrophils; (I, L) CD86⁺/F4/80⁺ and CD163⁺/F4/80⁺ macrophages. Bone-mets, bone-metastases; DC, dendritic cell; i.a., intra-arterial; MRI, magnetic resonance imaging; NK, natural kill; TAM, tumor-associated macrophage; TAN, tumor-associated neutrophil; ZA, zoledronic acid; αDKK1, DKK1 antibody. Data are presented as mean ± SEM. ns, not significant, *P < 0.05, **P < 0.01, and ***P < 0.001.