Abstract
Objective: Bone metastases are refractory to current therapies, primarily owing to the immunosuppressive metastasis tumor microenvironment (TME), which is dominated by myeloid cells. However, the function and regulatory mechanisms of myeloid compartments within the bone TME are incompletely understood. Herein we sought to delineate the role of the DKK1-CKAP4 axis in shaping myeloid cell-mediated immunosuppression in bone metastases and to identify potential therapeutic strategies targeting this pathway.
Methods: The composition and phenotypic characteristics of myeloid cells in the bone TME were analyzed. Mechanistic studies were conducted using ex vivo co-culture systems and an in vivo Ckap4fl/flS100a8Cre (neutrophil-specific Ckap4 knockout) mouse model to delineate the role of the DKK1-CKAP4 axis in regulating neutrophil maturation, osteoclast-like cell differentiation, and macrophage polarization. The therapeutic efficacy of DKK1 blockade in combination with zoledronic acid, as well as the impact on remodeling the TME, was further validated in bone metastasis mouse models.
Results: Significant expansion and predominance of myeloid cells within the bone TME was noted. Mechanistically, the DKK1-CKAP4 axis regulated the development and function of multiple myeloid populations. The DKK1-CKAP4 axis drove neutrophils toward an immature-like, immunosuppressive phenotype, promoted osteoclast-like cell differentiation, and induced macrophage polarization into an M2-like phenotype. Importantly, combined therapy with DKK1 blockade and zoledronic acid reprogrammed immunosuppressive myeloid cells, restored antitumor immunity, and significantly reduced tumor burden in bone metastasis models.
Conclusions: The findings herein showed the DKK1-CKAP4 axis to be a key regulator of myeloid-driven immunosuppression in the bone TME. The combination of DKK1 blockade with zoledronic acid represents a potential therapeutic strategy for bone metastases.
keywords
Introduction
Bone is a common site of metastasis for various solid tumors, including breast, prostate, and lung cancers1,2. Bone metastases are frequently accompanied by severe skeletal-related events (SREs), such as pathologic fractures, intractable bone pain, and hypercalcemia, which threaten the quality of life and survival of bone-metastatic cancer patients3,4. Despite the significant morbidity and mortality associated with bone metastases, traditional therapies, including chemotherapy and radiotherapy, provide limited clinical benefit when compared to the efficacy against primary tumors5. In current clinical practice, bone-targeted agents (BTAs), including bisphosphonates (e.g., zoledronic acid), are routinely used as standard therapy for patients with bone metastases6. These agents exert effects predominantly through suppression of osteoclast-dependent bone degradation, thereby preserving bone integrity and preventing SREs7–12. Immunotherapies, such as immune checkpoint blockade (ICB), have shown promising efficacy in various solid tumors. However, the therapeutic responses in bone metastases are unsatisfactory, owing to the unique bone metastases microenvironment13–17.
Myeloid cells are the predominant population in the bone metastatic niche, shaping a profoundly immunosuppressive microenvironment18. Among the myeloid cells, neutrophils constitute the largest myeloid subset within the bone marrow microenvironment. Tumor-associated neutrophils (TANs) have gained considerable attention for their dual roles in the tumor microenvironment (TME), where they mediate crosstalk between tumor cells and other immune components, such as T cells, macrophages, and dendritic cells (DCs), thereby significantly influencing the response to immunotherapy19,20. Our recent work has shown that immature TANs have a critical impact on bone metastasis progression by fostering an immunosuppressive microenvironment21. In addition, emerging evidence highlights the multiple pro-tumor functions of osteoclasts in bone metastases. Beyond the classical function in bone resorption and facilitating tumor colonization, osteoclasts can reshape both local and distant microenvironments to promote metastatic progression22–24. Moreover, macrophages are highly enriched in the bone TME, where macrophages exert immunosuppressive effects and drive therapeutic resistance through both microenvironmental regulation and direct interactions with tumor cells25,26. Despite these findings, the mechanisms underlying immune resistance in the bone TME are poorly understood. Therefore, it is imperative to further elucidate the function of myeloid cells in the bone metastasis microenvironment and to identify potential therapeutic targets.
In Part I the immune landscape of bone metastasis was characterized by CyTOF analysis of bone marrow CD45+ immune cells from tumor-bearing and healthy mice. The therapeutic efficacy of zoledronic acid was evaluated by measuring tumor burden and immune changes in the bone microenvironment. The bone TME was dominated by expanded immunosuppressive myeloid populations and zoledronic acid alone failed to effectively reprogram the myeloid compartment or suppress tumor progression. In Part II mechanisms underlying myeloid reprogramming was investigated using ex vivo co-culture assays, neutrophil-specific Ckap4 deletion mouse models, and integrative clinical database analyses, which showed that the DKK1-CKAP4 signaling axis impairs neutrophil maturation, promotes the differentiation of OCPs toward osteoclast-like cells, and drives macrophage polarization in the bone TME. In Part III the combination of DKK1 blockade and zoledronic acid was shown to remodel the bone metastatic immune microenvironment, inhibits tumor progression, and enhance antitumor immunity. CyTOF, mass cytometry; DKK1, Dickkopf-related protein 1; CKAP4, cytoskeleton-associated protein 4; OCP, osteoclast precursor cell. Figure created with Microsoft PowerPoint.
Dickkopf-1 (DKK1), a secreted glycoprotein that antagonizes Wnt/β-catenin signaling, exerts tumor-promoting and immunomodulatory functions by engaging its receptor cytoskeleton-associated protein 4 (CKAP4)27–29. CKAP4 is widely expressed on various tumor cells and is also prominently present on myeloid cells within the TME28,29. In multiple tumor contexts, elevated DKK1 binds to CKAP4 on tumor cells and promotes tumor cell migration and proliferation27,30–32. In addition to these tumor-intrinsic functions, CKAP4 expression on myeloid immune cells enables the DKK1-CKAP4 axis to modulate the TME by promoting the recruitment and polarization of immunosuppressive myeloid populations33–35. DKK1 is predominantly secreted by osteoblasts and osteocytes, resulting in high concentrations in the bone marrow niche21. Clinically, elevated DKK1 levels in tumors and serum are associated with bone metastases in breast, lung, and prostate cancers36–38. These studies suggested that DKK1-CKAP4 signaling may have a profound impact on the immune landscape during bone metastases. However, the specific role of the DKK1-CKAP4 axis in the bone metastasis microenvironment and its potential as a therapeutic target are unclear.
In this study the profound impact of myeloid populations in the bone TME was identified, including immature neutrophils, activated osteoclasts, and immunosuppressive macrophages. These myeloid populations are reprogrammed by the DKK1-CKAP4 axis toward a more immunosuppressive phenotype. Moreover, combining myeloid cell reprogramming with zoledronic acid exerts potent antitumor effects and reshapes the bone metastasis microenvironment (see Study Flowchart).
Materials and methods
Cell line
The 4T1 cell line, a triple-negative mammary carcinoma originating from Balb/c mice, and the Lewis lung carcinoma (LLC) cell line, a Lewis lung carcinoma originating from C57BL/6 mice, were acquired from the National Collection of Authenticated Cell Cultures (Shanghai, China). Both cell lines were cultured in RPMI-1640 complete medium (Gibco, Grand Island, NY, USA), as previously described21.
Mice
BALB/c and C57BL/6 mice were used in this study. These animals were acquired from the Institute of Hematology, Chinese Academy of Medical Sciences (Tianjin, China). C57BL/6JGpt-Ckap4em1Cflox/Gpt (Ckap4fl/fl) and Ckap4fl/flS100a8cre mice were provided by GemPharmatech Co., Ltd (Nanjing, China). Six- to eight-week-old female mice were randomly divided into experimental groups for in vivo experiments. The Institutional Animal Care and Use Committee of Drum Tower Hospital approved all animal procedures (approval No. 2020AE01064).
Human data analysis
Transcriptomic data of metastatic samples from breast cancer patients were obtained from the Gene Expression Omnibus (GEO) database [GSE14018 (n = 58), GSE14020 (n = 65), and GSE54323 (n = 18)]. DKK1 mRNA expression in bone metastases was compared to metastases at other sites. It should be noted that GSE14020 integrates samples originally derived from GSE14018 and GSE14017. Owing to the batch effect adjustment and normalization performed during data merging, DKK1 expression in GSE14020 may exhibit minor discrepancies compared to the original GSE14018 series. Gene set enrichment analysis (GSEA) was performed to explore the relationship between DKK1 expression and bone-related biological pathways. Spearman’s rank correlation was used to examine the associations between DKK1 and several immune- or myeloid-related markers (CCL20, CCL7, CXCL5, S100A8, S100A9, and TNFRSF14).
Single-cell RNA sequencing (scRNA-seq) data (GSE143791) were used. This dataset was comprised of the following bone marrow samples: bone metastatic prostate tumors (tumor site); uninvolved bone marrow (distal); and bone marrow from cancer-free orthopedic patients (benign). Quality control, normalization, and batch effect correction were performed following standard pipelines. Dimensionality reduction and cell clustering were performed using t-distributed stochastic neighbor embedding (t-SNE) to visualize the major immune cell subsets (B cell, myeloid cell, NK cell, plasma cell, and T cell) with the myeloid cell compartment, including monocytes, macrophages, neutrophils, osteoclasts, and DCs. CKAP4 expression was examined across all cells as well as within the myeloid cell subset and a feature plot was generated to illustrate the distribution of CKAP4 expression. Myeloid cells in the tumor site group were divided into CKAP4-high and -low subgroups based on CKAP4 expression. Signature scores were calculated for each subgroup and compared to the following gene sets: immunosuppression-related genes (LGALS9, LILRB3, LILRB2, HIF1A, CD44, CD47, PTPN2, TGIF1, and S100A8) and antigen presentation-related genes (HLA-DRA, HLA-DPB1, HLA-B, HLA-A, PSMB9, PSMB8, CTSS, CTSB, and CLEC7A).
In vivo experiments
Construction and treatment of bone metastases models
4T1 cells were resuspended in phosphate-buffered saline (PBS) (2 × 106 cells/mL) to establish the 4T1 bone metastasis model and 100 μL of the cell suspension was administered into the caudal artery (CA) of BALB/c mice. LLC cells were prepared in PBS (5 × 106 cells/mL) for the LLC bone metastases model and 100 μL of the suspension was administered into the CA of C57BL/6 mice.
After tumor inoculation, mice were randomly divided to receive intraperitoneal (i.p.) treatment with one of the following regimens: mouse monoclonal DKK1 antibody [αDKK1] (10 mg/kg once every 3 d; Leap Therapeutics, Cambridge, MA, USA); IgG2a isotype control [C1.18.4 (10 mg/kg once every 3 d); BioXCell, Lebanon, NH, USA]; zoledronic acid (100 μg/kg once weekly; Sigma, St. Louis, MO, USA); or a combination of αDKK1 and zoledronic acid. The body weights of mice from different groups were recorded during the treatment schedule.
Generation of Ckap4fl/flS100a8cre mice
The Ckap4fl/fl mouse line (C57BL/6JGpt-Ckap4em1Cflox/Gpt) was generated by (GemPharmatech, Nanjing, China). Ckap4flox/flox mice were crossed with a transgenic line expressing Cre recombinase under the control of the S100a8 promoter [C57BL/6JGpt-H11em1Cin(hS100a8-iCre)/Gpt; S100a8cre] to generate mice with neutrophil-specific deletion of Ckap4. Genotyping results confirming the expected recombination are presented in Figure S1.
Magnetic resonance imaging (MRI) analysis
Tumor burden was assessed by MRI on day 21 using a BioSpec 7T system (Bruker BioSpin, Billerica, MA, USA). Bone metastases in the hind limbs of mice treated with IgG2a, zoledronic acid, αDKK1, or the combination of αDKK1 and zoledronic acid were visualized and images were evaluated with RadiAnt DICOM Viewer (2020.1).
Histologic analysis
Mice were sacrificed at the experimental endpoint and the femurs were harvested. The bone samples were fixed, decalcified, dehydrated, embedded, and sectioned (5 μm), as previously described21. Bone lesions were then stained with hematoxylin and eosin (H&E) or tartrate-resistant acid phosphatase (TRAP). The number of TRAP-positive cells per millimeter of bone interphase was quantified using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA).
Flow cytometry analysis
Bone marrow from mice in each group was immediately collected at the experimental endpoint. Cells were harvested from the tibias and femurs by centrifugation, then passed through 40-μm nylon strainers (Biosharp, Hefei, China). Erythrocytes were depleted using red blood cell lysis buffer (Biosharp), followed by two additional PBS washes. Prior to surface marker staining, cells were subjected to viability dye staining (BioLegend, San Diego, CA, USA) and Fc receptor blocking (BioLegend) following recently published protocols39. Single-cell suspensions were stained with the corresponding mouse antibodies for 30 min at 4°C, then washed twice with PBS for detection of cell surface markers [CD3, CD8, CD11b, CD11c, F4/80, CD80, CD86, CD163, Gr-1, MHC-II, NKp46, CD25, CD69, CD107a, Ly6G, CD101, and CKAP4] (BioLegend). Intracellular staining for CD206, Arg1, and iNOS was performed using anti-mouse antibody in combination with the Fixation/Permeabilization Solution Kit (BD Biosciences, San Jose, CA, USA). Stained cells were analyzed on a BD Accuri C6 PLUS flow cytometer (BD Biosciences) and all raw data were analyzed with FlowJo software (version 10.4; Tree Star, Ashland, OR, USA).
Ex vivo experiments
Mass cytometry (CyTOF) analysis
Bone marrow samples were collected from 3 healthy BALB/c mice (6–8 weeks old) and 3 BALB/c mice bearing 4T1 bone metastases. Single-cell suspensions were prepared, followed by red blood cell lysis. CD45+ immune cells were subsequently isolated through fluorescence-activated cell sorting (FACS) and pooled into one sample per group. For each sample, approximately 3.5 × 105 CD45+ cells were sorted and submitted for CyTOF analysis (PLT TECH, Hangzhou, China). After data acquisition, a total of 232,232 CD45+ cells from healthy mice and 287,458 CD45+ cells from bone metastasis-bearing mice were retained for downstream analysis. Cells were stained using a panel of metal-conjugated antibodies targeting surface and intracellular markers, including CD45, CD3e, Ki67, CD117, MHC-II, Granzyme B, CX3CR1, CD163, CD38, Ly6G, Ly6C, CD19, CD127, CD62L, CD11c, CD44, CD24, CD103, Gr-1, FcεRIα, TCRγδ, F4/80, TCRβ, CD64, CD69, CD25, CD86, NK1.1, CD27, CD206, FOXP3, CD137, T-bet, and CD80. Following staining, cells were acquired on a CyTOF instrument.
Data were normalized and analyzed using established pipelines. Immune cell populations were identified and characterized based on canonical marker expression, enabling comprehensive profiling of lymphoid and myeloid compartments within the bone marrow microenvironment.
Neutrophil culture and treatment
Mouse immature neutrophils (CD11b+Ly6G+CD101−) were isolated from the bilateral femurs and tibias of Balb/c and C57BL/6 mice using FACS. The purified cells were cultured for 24 h with recombinant mouse DKK1 (50 ng/mL; Abcam, Cambridge, UK) and GM-CSF (50 ng/mL; PeproTech, Cranbury, NJ, USA). After incubation, the expression of maturation- and activation-associated markers, including Ly6G, CXCR2, CXCR4, and CD101, were assessed by flow cytometry on a Beckman CytoFLEX LX instrument (Beckman Coulter, Brea, CA, USA).
Neutrophil and CD8+ T cell co-culture
Immature neutrophils were isolated and cultured as described above. CD8+ T cells were obtained from the spleens of BALB/c and C57BL/6 mice using a CD8+ T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and subsequently activated ex vivo as previously described35. After 3–5 d of activation, neutrophils and CD8+ T cells were co-cultured in fresh medium at a 1:1 ratio (2 × 106 cells/mL each) for 48 h. Following co-culture, CD8+ T cells were re-isolated using the same MACS-based method and the activation features and cytotoxic function were assessed as described in the subsequent sections.
Immunosuppression assays and cytotoxicity assay of CD8+ T cells
After co-culture with neutrophils, CD8+ T cells were collected and the activation status was evaluated by flow cytometry using antibodies against mouse CD25, CD69, and CD107a. Cytotoxicity was assessed by co-culturing CD8+ T cells with tumor cells at an effector-to-target (E:T) ratio of 5:1, followed by CFSE/PI double staining (MedChemExpress, Monmouth Junction, NJ, USA) to evaluate cell viability, as previously described35. The percentage of apoptotic cells was calculated according to the following standard formula: 100 × (PI+ CFSE+ cells)/(total CFSE+ cells) %. In addition, culture supernatants collected after the 8 h co-incubation were used for cytokine measurement, as described below.
Osteoclast induction
Bone marrow isolated from the femurs and tibias of BALB/c mice was flushed with cold DMEM (Gibco) and made into a red blood cell-free single-cell suspension. Cells were cultured with M-CSF and RANKL (R&D Systems, Minneapolis, MN, USA) to induce osteoclast precursor cells (OCPs), as described in a previous report40. After 7 d cells were treated with 50 ng/mL of recombinant mouse DKK1 or a mixture of DKK1 and 10 μg/mL of αDKK1 for an additional 48 h. Osteoclast-like cells were stained with a TRAP staining kit (Servicebio, Wuhan, China). TRAP+ cell density (cells/mm) was quantified using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA). The identification of TRAP+ multinucleated osteoclast-like cells was independently confirmed by an experienced pathologist based on morphologic criteria (≥3 nuclei and strong TRAP staining).
Macrophage induction
Bone marrow cells were harvested from the bilateral femurs and tibias of BALB/c and C57BL/6 mice, collected by centrifugation, and seeded into 12-well plates at a density of 1 × 106 cells/mL in medium supplemented with 10% FBS (Gibco) and 50 ng/mL of M-CSF (PeproTech). The medium was refreshed on day 3 and macrophage differentiation was confirmed by flow cytometry on day 5.
Ex vivo mouse macrophage polarization and treatment
Bone marrow-derived macrophages (BMDMs) from BALB/c or C57BL/6 mice were seeded at 3 × 105 cells per well in 12-well plates. Cells were left untreated as controls or polarized to an M1 phenotype with LPS (50 ng/mL; Sigma-Aldrich) for 48 h. Cells in separate groups were pre-incubated with recombinant mouse DKK1 (50 ng/mL), DKK1 + αDKK1 (10 μg/mL), or DKK1 + αCKAP4 [10 μg/mL (3F11-2B10, provided by Professor Akira Kikuchi from Osaka University, Osaka Prefecture, Japan)] for 48 h. After stimulation, cells were harvested and washed with PBS. Flow cytometry was performed to detect surface markers (CD45, CD11b, F4/80, CD80, CD86, and CD163) and intracellular markers (CD206, iNOS, and Arg1), as described above.
Ex vivo mouse myeloid cell culture and polarization
One week after tumor inoculation, CD11b+ myeloid cells were isolated from the bone marrow of 4T1 bone metastasis-bearing BALB/c mice using anti-mouse CD11b magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany). Purified CD11b+ cells were seeded in 12-well plates at a density of 5 × 105 cells per well in RPMI-1640 complete medium. Cells were then stimulated with lipopolysaccharide [LPS] (50 ng/mL; Sigma-Aldrich) alone or in combination with recombinant mouse DKK1 (50 ng/mL; Abcam), DKK1 + αDKK1 [10 μg/mL; provided by (Leap Therapeutics, Cambridge, MA, USA)], or DKK1 + αCKAP4 [10 μg/mL, (3F11-2B10, provided by Professor Akira Kikuchi from Osaka University, Osaka Prefecture, Japan)] for 48 h at 37°C in a 5% CO2 incubator. After stimulation, cells were harvested and washed twice with PBS. Flow cytometry was performed to detect surface markers (CD45, CD11b, F4/80, CD80, CD86, CD163, Ly6G, CD101, and CD61) and intracellular markers (CD206), as described above.
Cytometric bead array (CBA) analysis
The concentrations of IFN-γ, IL-6, TNF-α, CCL2, and IL-10 in cell culture supernatants were measured using the CBA Mouse IFN-γ Flex Set and the CBA Mouse Inflammation Kit (BD Biosciences). Sample acquisition was performed on a flow cytometer (Beckman CytoFLEX LX).
Statistical analysis
All statistical analyses and data visualization were performed using GraphPad Prism 10 (GraphPad Software, San Diego, CA, USA). The two-tailed Student’s t-test was applied for two-group comparisons. One-way ANOVA was used for comparisons among three or more groups. All values are shown as the mean ± SEM. Statistical significance was set at a P < 0.05. Significance is denoted as ns (not significant), *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.
Results
The bone metastasis microenvironment is predominantly composed of immunosuppressive myeloid cell populations
A 4T1 syngeneic bone metastasis model was established using the previously reported intra-caudal artery (CA) injection method to determine the immune landscape of the bone metastasis microenvironment41. Bone marrow samples were harvested from tumor-free and bone metastasis-bearing mice, and CD45+ immune cells were isolated for high-dimensional immune profiling using CyTOF. Unbiased hierarchical clustering combined with supervised annotation identified 26 distinct immune cell populations, including monocytes/macrophages (Mono/Mac), neutrophils, DCs, eosinophils, basophils, B cells, CD4+ T cells, CD8+ T cells, and NK cells (Figure 1A–C). Bone metastasis-bearing mice exhibited a markedly altered bone marrow immune landscape that was characterized by prominent expansion of myeloid subsets, particularly Mono/Mac and neutrophils, together with a notable reduction in DCs compared to tumor-free mice (Figure 1D). In addition, the frequencies of effector lymphocyte populations, including B cells, CD4+ T cells, CD8+ T cells, and NK cells, were significantly decreased in the bone metastasis group (Figure 1E). Interestingly, eosinophils were markedly enriched in the bone marrow of metastatic mice, whereas basophil frequencies remained unchanged (Figure 1E), indicating a specific remodeling of granulocyte populations during bone metastatic progression.
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.
Zoledronic acid, a standard treatment for patients with bone metastases, was administered once each week to 4T1 bone metastasis-bearing mice and the tumor burden and immune alterations within the bone TME were evaluated (Figure 1F). Direct observation and micro-MRI demonstrated that zoledronic acid treatment failed to suppress bone metastasis growth (Figure 1G, H). Flow cytometry analysis of the bone marrow immune microenvironment showed no significant changes in the abundance of CD8+ T cells, NK cells, or DCs following treatment (Figure 1I–K). Although the total proportion of macrophages was not significantly changed, zoledronic acid treatment significantly promoted a phenotypic shift toward an M1-like state (Figure 1L, M), indicating partial alleviation of the immunosuppressive macrophage phenotype. However, neutrophil frequency and maturation status remained unchanged after treatment (Figure 1N), suggesting that zoledronic acid alone provides only limited modulation of the myeloid compartment, which is insufficient to elicit an antitumor response in bone metastases.
DKK1 impairs neutrophil maturation to induce immunosuppressive function
Given the pivotal roles of myeloid cells, including neutrophils, osteoclasts, and macrophages within the bone TME, elucidating the regulatory mechanisms underlying cellular differentiation and function is essential for developing effective therapies. In our recent study we identified a significant upregulation of DKK1 in bone metastases compared to primary tumors21, suggesting a potential role in shaping the immunosuppressive bone metastasis microenvironment. To further define how DKK1 signaling influences the phenotype and function of these key myeloid subsets, a series of comprehensive ex vivo functional assays were performed. Immature neutrophils have been increasingly recognized as key mediators of immunosuppression within the TME. Given the numerical abundance and immunoregulatory potential in the bone marrow, we sought to investigate whether DKK1 contributes to immune evasion by modulating neutrophil maturation. Immature neutrophils (Ly6G+CD101−) were first isolated from the bone marrow of healthy C57BL/6 mice by flow cytometry and subjected to ex vivo differentiation assays. Upon GM-CSF stimulation, these neutrophils progressively acquired phenotypic markers of maturation, including upregulation of Ly6G, CXCR2, and CD101, along with downregulation of CXCR4 (Figure 2A). However, the addition of recombinant mouse DKK1 protein significantly impaired GM-CSF-induced neutrophil maturation (Figure 2A), indicating that DKK1 has a crucial role in maintaining the immature state of neutrophils.
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.
To evaluate the immunological consequences of this impaired maturation, flow-sorted immature neutrophils were treated with GM-CSF alone or in combination with DKK1 for 24 h followed by co-culture with magnetically isolated splenic CD8+ T cells (Figure 2B). Functional assays revealed that immature neutrophils profoundly suppress CD8+ T-cell activation, while GM-CSF-induced maturation markedly attenuates the immunosuppressive capacity (Figure 2C–E). Notably, DKK1 exposure restored the immunosuppressive phenotype of these neutrophils, as evidenced by decreased IFN-γ production (Figure 2C), reduced expression of CD8+ T-cell activation markers [CD25, CD69, and CD107a] (Figure 2D), and impaired cytotoxicity against LLC tumor cells (Figure 2E). Collectively, these findings identified DKK1 as a critical regulator of neutrophil maturation, sustaining an immunosuppressive state of immature neutrophils that compromises CD8+ T-cell effector functions ex vivo.
Neutrophil-specific Ckap4 deletion promotes neutrophil maturation and restrains bone metastatic tumor progression
CKAP4 is a transmembrane receptor that was recently identified as a functional binding partner of DKK127. CKAP4 transduces downstream signaling that regulates cellular functions, such as proliferation, differentiation, and immune modulation by directly interacting with DKK1. This finding suggests that binding of DKK1 to CKAP4 on TANs may impair neutrophil maturation and alter the functional properties, which contributes to tumor-promoting effects (Figure 3A). Neutrophil-specific Ckap4 knockout mice (Ckap4fl/flS100a8Cre) were generated to further elucidate the role of CKAP4 in regulating neutrophil maturation and the impact on tumor progression (Figures 3B and S1). This model enables neutrophil-specific gene deletion with high efficiency and minimal off-target recombination in other myeloid compartments. Bone metastasis models were established by CA injection of LLC cells into a Ckap4fl/fl and Ckap4fl/flS100a8Cre mouse model with experimental endpoints set at day 28. Direct observation and micro-MRI imaging revealed that deletion of Ckap4 in neutrophils significantly suppresses bone metastatic tumor growth compared to the Ckap4fl/fl control group (Figure 3C, D).
Neutrophil-specific Ckap4 deletion promotes neutrophil maturation and restrains bone metastases tumor progression. (A) Schematic illustrating DKK1-CKAP4 interaction on neutrophils. (B, C) Ckap4fl/fl and Ckap4fl/flS100a8cre 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.
Bone marrow samples were harvested to investigate changes in the bone metastasis microenvironment at the endpoint for analysis. Flow cytometry confirmed enhanced neutrophil maturation in the bone marrow of Ckap4fl/flS100a8Cre mice (Figure 3E). Analysis of myeloid cells revealed a substantial increase in M1-like macrophages (CD86+F4/80+), a reduction in M2-like macrophages (CD163+F4/80+), and increased activation of DCs (Figure 3F, G). Importantly, both the frequency and activation status of CD8+ T cells were significantly elevated in Ckap4-deficient mice compared to controls (Figure 3H, I). Collectively, these results demonstrated that CKAP4 acts as a key receptor mediating the immunosuppressive effects of DKK1 on neutrophil maturation. Inhibition of DKK1-CKAP4 signaling in neutrophils restores maturation and remodels the immunosuppressive microenvironment, thereby enhancing antitumor immune responses and restraining bone metastatic tumor progression.
DKK1 promotes the differentiation of OCPs into osteoclast-like cells
Bone marrow cells from healthy mice were induced into OCPs ex vivo with M-CSF and RANKL stimulation to investigate the regulatory role of DKK1 in osteoclast differentiation (Figure 4A). After 7 days of induction recombinant DKK1 protein or DKK1 combined with αDKK1 was added for an additional 48<nonbrfixspace>h co-culture. TRAP staining confirmed that DKK1 stimulation significantly increased the number of TRAP+ multinucleated osteoclast-like cells, while the addition of αDKK1 markedly attenuated this increase (Figure 4B, C), suggesting that DKK1 may promote the differentiation of OCPs toward an osteoclast-like phenotype. Furthermore, the functional activity of these osteoclast-like cells was evaluated by measuring the secretion of inflammatory cytokines, including IL-6, CCL2, and TNF-α. Osteoclast-like cells exhibited enhanced proinflammatory cytokine production following DKK1 co-culture, whereas the addition of αDKK1 significantly attenuated this effect (Figure 4D). These findings suggested that DKK1 enhances proinflammatory activity, which may further contribute to the immunosuppressive and osteolytic features of the tumor-bone microenvironment in addition to promoting differentiation of OCPs into osteoclast-like cells.
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.
GSEA was performed to validate these results in clinical datasets using transcriptomic datasets from patients with breast cancer (GSE14018, GSE14020, and GSE54323). The results revealed a significant positive correlation between DKK1 expression and osteoclast-related gene signatures, including pathways related to osteoclast differentiation, bone resorption, osteopenia, and cancer-related bone pain. Conversely, DKK1 expression was negatively correlated with osteoblast differentiation, ossification, bone remodeling, and BMP signaling pathways (Figure 4E). Collectively, these results support a role for DKK1 in promoting osteoclast-like differentiation and inflammatory activity, thereby potentially contributing to osteolytic bone destruction.
DKK1 promotes M2-like polarization and immunosuppressive function of macrophages
Macrophage polarization and functional status have important roles in tumor initiation and progression42,43. Ex vivo stimulation assays were performed to further explore the role of DKK1 in macrophage polarization using BMDMs. Bone marrow cells isolated from healthy BALB/c and C57BL/6 mice were cultured with M-CSF for 5 d to generate M0 macrophages. These cells were then stimulated with LPS to induce classical M1 polarization, then treated with recombinant DKK1 protein, DKK1 plus αDKK1, or DKK1 plus αCKAP4 (Figure 5A, E). Cytokine production by macrophages was first examined under different stimulation conditions. Stimulation with recombinant DKK1 protein in BALB/c-derived BMDMs led to significant upregulation of the immunosuppressive chemokine, CCL2, while this effect was markedly reversed upon co-treatment with αDKK1 (Figure 5B). DKK1 stimulation in C57BL/6-derived BMDMs resulted in a notable reduction in pro-inflammatory cytokines (IL-6 and TNF-α) accompanied by increased secretion of CCL2 and the anti-inflammatory cytokine, IL-10 (Figure 5F). Again, these effects were effectively reversed by DKK1 blockade with αDKK1, which restored a pro-inflammatory cytokine profile (Figure 5B, F).
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.
Consistent with these cytokine profiles, flow cytometric analysis revealed that recombinant DKK1 protein markedly suppressed LPS-induced M1 polarization, as evidenced by a reduced percentage of CD80+CD86+ macrophages (Figure 5C, G). Conversely, DKK1 concurrently promoted the expression of M2-associated markers, reflected by an increased proportion of CD163+CD206+ macrophages (Figure 5D, H), indicating that DKK1 skews macrophage polarization toward an immunosuppressive M2-like phenotype. Co-treatment with αDKK1 or αCKAP4 partially reversed the suppressive effects of DKK1, restoring M1-associated marker expression and reducing the acquisition of M2 features (Figure 5C, D, G, H). In support of these experimental findings, integrative transcriptomic analyses of human breast cancer databases (GSE14018, GSE14020, and GSE54323) showed that DKK1 expression is positively correlated with myeloid-derived chemokines, such as CCL20, CCL7, and CXCL5, as well as pro-tumor neutrophil markers, like S100A8 and S100A9 (Figure 5I). In parallel, DKK1 expression negatively correlated with TNFRSF14, a known co-stimulatory molecule involved in T-cell activation (Figure 5I). Together, these results indicated that DKK1 promotes macrophage-mediated immunosuppression by skewing macrophage polarization toward the M2 phenotype, thereby fostering the formation of an immunosuppressive TME conducive to tumor progression.
The DKK1-CKAP4 axis globally reprograms multiple myeloid cell populations in the bone metastasis microenvironment
A 4T1 bone metastasis model was established to investigate the impact of the DKK1-CKAP4 axis on global myeloid cell populations within the bone metastasis microenvironment. CD11b+ myeloid cells were isolated from the bone marrow 7 d after tumor inoculation and cultured ex vivo in the presence of LPS with or without recombinant DKK1 protein, αDKK1, or αCKAP4 (Figure S2A). After 48 h flow cytometric analysis revealed that co-culture with DKK1 significantly reduced CD101 expression on neutrophils (Figure S2B), increased the proportion of CD61+ osteoclast-like cells (Figure S2C), decreased the frequency of CD80+CD86+ M1-like macrophages (Figure S2D), and elevated the percentage of CD163+CD206+ M2-like macrophages (Figure S2E). Conversely, co-treatment of αDKK1 or αCKAP4 restored CD101 expression on neutrophils, reduced the proportion of CD61+ osteoclast-like cells, increased M1-like macrophages, and decreased M2-like macrophages (Figure S2B–E). Of note, CD61 has been reported to be expressed not only on osteoclasts but also on megakaryocytes and platelets44,45. In our study CD11b+ myeloid cells were positively selected prior to differentiation, which effectively excluded megakaryocytes and platelets. Therefore, an increase in CD61 expression may indicate enhanced differentiation of OCPs toward an osteoclast-like phenotype. These findings indicated that DKK1, acting through CKAP4, globally reprograms multiple myeloid lineages toward an immunosuppressive phenotype. Blockade of DKK1-CKAP4 signaling effectively reverses this myeloid-suppressive program.
Single-cell RNA sequencing data from the GSE143791 dataset were analyzed to investigate the role of the DKK1-CKAP4 pathway in the human bone metastatic tumor microenvironment, which includes samples from bone metastatic prostate tumors (tumor site), uninvolved bone marrow (distal), and bone marrow from cancer-free orthopedic patients (benign). Dimensionality reduction and clustering using t-SNE were performed to visualize immune cell populations (Figure S3A, B). CKAP4 expression was then examined across different immune cell subsets, which showed that CKAP4 was predominantly expressed in myeloid cells (Figure S3C). Analysis of CKAP4 expression levels in myeloid cells across the different sample groups revealed that CKAP4 was primarily expressed in the tumor site group (Figure S3D). Myeloid cells in the tumor site were stratified into CKAP4-high and -low subgroups based on CKAP4 expression. Comparison of gene signatures between the two subgroups showed that the CKAP4-high subgroup exhibited elevated expression of immunosuppressive-related genes, including LGALS9, LILRB3, LILRB2, HIF1A, CD44, CD47, PTPN2, TGIF1, and S100A8 (Figure S3E). In contrast, the CKAP4-low subgroup displayed higher expression of antigen-presentation-related genes, such as HLA-DRA, HLA-DPB1, HLA-B, HLA-A, PSMB9, PSMB8, CTSS, CTSB, and CLEC7A (Figure S3F). Collectively, these findings indicated that CKAP4-high myeloid cells possess a more immunosuppressive phenotype, while CKAP4-low myeloid cells exhibit features consistent with immune activation and enhanced antigen presentation.
Combining myeloid cell reprogramming with zoledronic acid suppresses tumor progression and remodels the bone metastasis immune microenvironment
Given that DKK1 promotes osteoclast-like cell differentiation and the immunosuppressive functions of myeloid cells, we hypothesized that blockade of DKK1-CKAP4 signaling with αDKK1 in combination with zoledronic acid may exert potent antitumor effects. To test this hypothesis, a 4T1 bone metastasis model in BALB/c mice and an LLC bone metastasis model in C57BL/6 mice were separately established. Mice were treated with an αDKK1 or IgG2a isotype control intraperitoneally twice weekly, while zoledronic acid was administered intraperitoneally on days 7 and 15. The experimental endpoint was assessed on day 21 (Figure 6A, D). Direct observation and micro-MRI imaging demonstrated that αDKK1 treatment significantly suppressed tumor progression in both models without evident weight loss and the combination therapy produced a markedly greater reduction in tumor burden compared with αDKK1 monotherapy, supporting a synergistic antitumor effect (Figures 6B, C, E, F and S4C, H). H&E and TRAP staining was performed on bone metastasis sections in the 4T1 bone metastasis model. H&E staining confirmed that zoledronic acid alone failed to control bone metastatic progression, whereas αDKK1 treatment significantly reduced tumor infiltration with combination therapy showing much more pronounced effects. Moreover, zoledronic acid monotherapy decreased the number of osteoclasts and combination therapy led to a greater reduction (Figure S4A, B).
![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.](https://www.cancerbiomed.org/content/cbm/early/2026/07/27/j.issn.2095-3941.2026.0168/F7/graphic-14.large.jpg?width=800&height=600&carousel=1)
![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.](https://www.cancerbiomed.org/content/cbm/early/2026/07/27/j.issn.2095-3941.2026.0168/F7/graphic-15.large.jpg?width=800&height=600&carousel=1)
![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.](https://www.cancerbiomed.org/content/cbm/early/2026/07/27/j.issn.2095-3941.2026.0168/F7/graphic-16.large.jpg?width=800&height=600&carousel=1)
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.
The composition and phenotypes of immune cells in the bone marrow of 4T1 and LLC bone metastases mice were analyzed to explore the alterations in immune competence within the bone TME following treatment (Figures 6G–L and S4D–G, I–L). DKK1 blockade alone substantially increased the frequency and activation of CD8+ T cells (Figure 6G, J), enhanced the infiltration of NK cells and MHC-II+ DCs, and reduced the accumulation of Arg1+ myeloid-derived suppressor cells [MDSCs] (Figure S4D–F, I–K). Although the overall frequency of Ly6G+ neutrophils was not significantly altered, αDKK1 treatment promoted neutrophil maturation, as evidenced by increased expression of the maturation marker CD101 (Figure 6H, K). Moreover, DKK1 blockade reprogrammed macrophage polarization by increasing M1-like macrophages (CD86+) and decreasing M2-like macrophages (CD163+) (Figures 6I, L and S4G, L). Notably, administration of αDKK1 in healthy, tumor-free mice did not alter the composition or activation status of immune cell subsets in the bone marrow (Figure S5), indicating that the immunomodulatory effects are specific to the tumor-bearing context.
Combination treatment induced more pronounced microenvironment remodeling compared to DKK1 blockade alone. Specifically, combination treatment further increased the proportion and activation of CD8+ T cells in the 4T1 bone metastasis model (Figure 6G), whereas CD8+ T cell activation was enhanced without changes in the proportion in the LLC bone metastasis mice model (Figure 6J). NK cell infiltration remained unchanged in both models (Figure S4D, I). Furthermore, analysis of the myeloid compartment showed no significant increase in MDSC or DC infiltration (Figure S4E, F, J, K). The proportion of CD101+ mature neutrophils was significantly increased following combination treatment (Figure 6H, K). Importantly, a more pronounced shift was noted toward M1-like macrophage polarization that was accompanied by a concomitant reduction in M2-like macrophages (Figures 6I, L and S4G, L). Together, these findings demonstrated that combining myeloid cell reprogramming with zoledronic acid exerts potent antitumor effects in bone metastasis models by alleviating immune suppression, thereby offering a promising therapeutic strategy for the treatment of bone metastases.
Discussion
In this study myeloid cells within the bone metastasis microenvironment were shown to be crucial for establishing a profoundly immunosuppressive niche. Mechanistically, DKK1-CKAP4 signaling drives neutrophils toward an immature-like functional state, thereby contributing to immunosuppression. DKK1-CKAP4 signaling also promotes osteoclast-like cell differentiation, leading to increased bone destruction. In addition, DKK1-CKAP4 signaling drives polarization of TAMs toward an M2-like state, further reinforcing local immunosuppression. Given previous evidence that DKK1–CKAP4 signaling promotes tumor cell proliferation and survival through activation of the PI3K–AKT pathway, we hypothesize that this pathway may likewise contribute to DKK1–CKAP4-mediated immunosuppressive reprogramming of myeloid cells in the bone TME. Notably, myeloid cell reprogramming combined with zoledronic acid significantly suppressed bone metastasis progression, enhanced CD8+ T cell infiltration and activation, and promoted neutrophil maturation as well as M1 macrophage polarization in the bone TME (Figure 7).
Schematic models illustrating the reprogramming of myeloid cells in the bone metastasis microenvironment combined with zoledronic acid treatment. (A, B) Overall landscape and therapeutic impact. (A) Bone metastases are enriched with immunosuppressive myeloid cell populations, including neutrophils, macrophages, and osteoclasts, which all contribute to the formation of a tumor-promoting microenvironment. These findings provide a more in-depth understanding of the impact of myeloid cells within the bone metastasis microenvironment. (B) Therapeutic blockade of DKK1 combined with zoledronic acid reshapes the bone metastasis microenvironment, enhances antitumor immune responses, and suppresses tumor progression. These findings provide a potential strategy for bone metastasis treatment and broaden the application of zoledronic acid beyond bone protection. (C, D) Mechanism explanation. (C) Our results derived from neutrophil-specific Ckap4 knockout mice and ex vivo assays demonstrated that the DKK1-CKAP4 signaling axis drives the immunosuppressive and pro-tumorigenic functional states of myeloid cells in the bone metastasis microenvironment, including an immature-like neutrophil state, the differentiation of OCPs toward osteoclast-like cells, and M2-like macrophage polarization. Mechanistically, we hypothesize that DKK1-CKAP4 signaling activates the downstream PI3K–AKT pathway, thereby reinforcing myeloid-mediated immunosuppression, impairing CD8+ T-cell function, and facilitating immune evasion. (D) DKK1 blockade plus zoledronic acid could inhibit the DKK1-CKAP4-PI3K-AKT signaling axis, reprogram myeloid cell function, re-activate CD8+ T cells, and restore antitumor immunity. These findings uncover the underlying mechanism modulating myeloid cell function within the bone metastasis microenvironment and provide the mechanistic rationale for the combination therapy of DKK1 blockade and zoledronic acid in bone-metastatic patients. CKAP4, cytoskeleton-associated protein 4; DKK1, dickkopf-1. Figure created with BioRender.com.
Bone metastases exhibit a distinct microenvironment enriched with immunosuppressive myeloid cells, including TANs, osteoclasts and TAMs, which collectively drive tumor progression and therapeutic resistance compared to primary tumor sites18. The immune status of neutrophils in bone marrow has a profound impact on tumor growth, metastasis, and response to immunotherapy19,20. Extensive studies have focused on the maturation state of TANs, revealing that immature and mature neutrophils may exert heterogeneous functions depending on tumor type and metastasis site. Multi-omics studies and our previous work have shown that the bone metastasis microenvironment is enriched in bone metastasis-bearing mice with Ly6Glow CD101− immature neutrophils, which exhibit strong immunosuppressive activity, whereas CD101+ mature neutrophils in the bone TME do not suppress CD8+ T cell function21,46–48. Similar populations of immature neutrophils are also enriched in human cancers both (circulation and tumor sites) and promote immune evasion48–52. CXCR2 is a key driver of neutrophil maturation and function in mice. Cxcr2 deficiency reduces the proportion of mature neutrophils in the spleen and increases the number of CD62Llo CXCR4hi senescent neutrophils53. Many solid tumors upregulate CXCL12 and exploit the CXCL12-CXCR4 axis to recruit pathologically activated, immature-like neutrophils, while impairing T cell recruitment, thereby creating an immunosuppressive microenvironment54. Consequently, changes in CXCR2 and CXCR4 expression are often used as indicators of neutrophil maturation status. In addition to the immunosuppressive effects mediated by TANs, osteoclasts have a fundamental role in the “vicious cycle,” a well-established mechanism underlying bone metastasis55–57. A range of osteolysis regulators released by tumor cells, such as RANKL, can activate osteoclasts, which further accelerate bone destruction and release various cytokines, like transforming growth factor-beta (TGF-β), interleukin (IL)-19, and osteopontin (OPN), to promote bone metastases growth58,59. Moreover, previous studies have also highlighted the functional heterogeneity of TAMs within the bone metastasis microenvironment. For example, Ma et al. showed that TAMs in breast cancer bone metastases facilitate tumor growth through CCL2-CCR2 and IL4R signaling pathways25. Similarly, Li et al. demonstrated that in prostate cancer bone metastases, macrophage-derived activin-A activates the FN1-ITGβ5-SRC axis in tumor cells, contributing to androgen deprivation therapy resistance26. Our findings integrate these seemingly distinct observations by identifying DKK1-CKAP4 signaling as a key upstream regulator of this myeloid suppressive network. Rather than acting on a single cell type, DKK1 drives neutrophils toward an immature, immunosuppressive phenotype, promotes the differentiation of OCPs toward osteoclast-like cells, and induces macrophage polarization towards an M2-like state. Through these coordinated effects, DKK1 links immune suppression with bone destruction in the bone metastasis microenvironment. This mechanism helps explain how immunosuppression and osteolysis are simultaneously sustained in bone metastases.
As a classic inhibitor of the Wnt signaling pathway, DKK1 competitively binds to the LRP5/6 co-receptors and antagonizes Wnt/β-catenin signaling60. By disrupting this pathway, DKK1 contributes to tumor progression, in part by sustaining a poorly differentiated cellular state, which increases malignant behavior61. In parallel, DKK1 has also been shown to promote the recruitment and immunosuppressive functions of MDSCs through the same signaling axis, further supporting tumor growth in mouse models33. In addition to these canonical effects, recent studies have identified CKAP4 as a non-canonical receptor for DKK1. DKK1-CKAP4 signaling has been implicated in promoting tumor immune evasion at primary tumor sites by directly acting on tumor cells, where DKK1-CKAP4 signaling activates oncogenic pathways, such as PI3K/AKT, and enhances invasive capacity through integrin-mediated adhesion and downstream signaling pathways27,30,33. In addition to these tumor-intrinsic effects, accumulating evidence suggests that DKK1 also directly modulates immune cells. Notably, a recent study demonstrated that DKK1 impairs CD8+ T cell-mediated antitumor responses via the GSK3β/E2F1/T-bet axis in colorectal cancer models62, supporting a direct suppressive role of DKK1 on cytotoxic T cells. Our previous findings indicated that the immunosuppressive functions of DKK1 extend beyond direct effects on CD8+ T cells because CD8+ T cell depletion fails to fully abrogate the antitumor efficacy of DKK1 blockade in gastric cancer models35. Indeed, DKK1-CKAP4 signaling has been shown to promote M2-like polarization of TAMs in primary tumors, thereby indirectly dampening CD8+ T cell responses35. Moreover, our recent study demonstrated that DKK1–CKAP4 signaling activates the PI3K–AKT pathway in neutrophils, thereby promoting the immunosuppressive functional state21. Building on this finding, the present study further reveals that DKK1-CKAP4 signaling broadly reprograms multiple myeloid populations in the bone TME, including TANs, osteoclasts, and TAMs, potentially through activation of the PI3K–AKT pathway, collectively establishing an immunosuppressive microenvironment that constrains CD8+ T cell function. Together, these findings indicate that DKK1 exerts pleiotropic pro-tumor effects by coordinating tumor-intrinsic signaling, directly suppressing cytotoxic T cell activity, and indirectly impairing antitumor immunity through extensive reprogramming of the myeloid compartment, underscoring the potential as a target for combination immunotherapy strategies.
Clinically, accumulating evidence indicates a strong association between DKK1 expression and bone metastasis. A recent study by Zhuang et al. demonstrated that tumor-secreted DKK1 can regulate the organotropism of breast cancer metastasis with high DKK1-expressing tumors preferentially metastasizing to bone, while low DKK1-expressing tumors are more likely to metastasize to the lung63. Multiple retrospective studies have further reported that patients with bone metastases from breast, lung, or prostate cancers exhibit significantly elevated DKK1 levels in serum or tumor tissues compared to patients who only have primary tumors36–38. Osteoblasts and osteocytes are the most important physiologic source of DKK1, resulting in significantly higher DKK1 concentrations in the bone metastatic microenvironment than in most primary tumor sites64. The bone metastatic microenvironment exhibits a distinct cellular composition, characterized by aberrant expansion of myeloid cells, including massive infiltration of immunosuppressive neutrophils, osteoclasts, and M2-like macrophages. Therefore, the DKK1-CKAP4 signaling axis may have a more critical role in the bone TME compared to primary tumors. The single-cell dataset, GSE143791, was analyzed to further explore the clinical relevance of CKAP4 in the bone metastasis microenvironment. Notably, CKAP4 expression was markedly elevated in tumor site samples compared to uninvolved or benign bone marrow and this expression was predominantly restricted to myeloid cell populations. Moreover, CKAP4-high myeloid cells exhibited an immunosuppressive signature, which is characterized by high expression of inhibitory molecules (e.g., LGALS9, LILRB3, and S100A8) with low expression of antigen presentation signatures compared to CKAP4-low myeloid cells. These human data are consistent with our experimental findings in mouse models. Building on these findings, our data demonstrated that DKK1-CKAP4 signaling is also essential for shaping the bone metastasis microenvironment. Specifically, this axis regulates myeloid cell differentiation and function, thereby promoting a sustained immunosuppressive niche. Together, these results indicated that DKK1-CKAP4 signaling not only drives primary tumor progression but also actively contributes to the establishment of the bone metastasis environment.
Despite the availability of multiple therapeutic strategies for primary tumors65,66, effective treatments for bone metastases are limited. Chemotherapy, radiotherapy, and immunotherapies generally elicit unsatisfactory responses in this setting13,15,17. Given the profoundly immunosuppressive microenvironment of bone metastases, monotherapy is often insufficient, making combination therapy particularly important. Recent studies have demonstrated progress in the treatment of bone metastases using conventional bone-protective agents and myeloid reprogramming therapies. For example, osteoclast-derived OPN mediates systemic resistance to ICB and combining ICB with an anti-RANKL antibody effectively blocks osteoclastogenesis and restores ICB efficacy24. Strategies co-targeting T cells and myeloid cells have also made important progress. For example, concurrent blockade of TIGIT on T cells and IL-1β produced by myeloid cells in a breast cancer bone metastasis model effectively activated antitumor immunity, suppressed bone metastases, and improved survival67. These studies indicated that conventional bone-protective agents possess therapeutic potential and targeting myeloid cells represents a critical component of immunotherapy within the bone metastatic microenvironment. Zoledronic acid, as another commonly used bone-protective agent, is a standard treatment for patients with bone metastases and is widely used in clinical practice to prevent SREs12. However, the impact on overall survival remains limited, highlighting the need for more effective combination therapies7,9,68–70. In addition to bone-protective effects, zoledronic acid has been reported to exert modest immunomodulatory activity. Preclinical studies have shown that zoledronic acid partially induces M1-like polarization of macrophages and suppresses M2-associated markers, suggesting a potential role in modulating the TME71. Nevertheless, these effects appear insufficient to reverse the profound immunosuppression observed in bone metastases, suggesting that combining zoledronic acid with myeloid reprogramming may hold therapeutic promise. In this study we found that combining myeloid reprogramming with zoledronic acid markedly enhanced the recruitment and activation of CD8+ T cells, drove neutrophil maturation, and promoted macrophage polarization toward the M1 phenotype within the TME, thereby exerting potent antitumor effects. Notably, the DKK1-neutralizing antibody, DKN-01, has already shown encouraging clinical activity in primary tumor sites72,73, supporting the translational potential of this combination strategy for patients with bone metastases. Taken together, our findings demonstrated that zoledronic acid, when combined with myeloid reprogramming, functions not only as a bone-protective agent but also an effective drug for tumor control. This combination markedly improves the antitumor activity of a widely used clinical drug and extends the role from preventing skeletal-related events to actively suppressing tumor progression in bone metastases.
Our study had several limitations. First, we only used two syngeneic murine bone metastases models (4T1 and LLC), which may not fully reflect the heterogeneity of metastatic disease across diverse tumor types. Second, our findings have not been validated in clinical samples, so corollary studies should include analyses of bone metastasis from patients to confirm the translational relevance of these findings. In addition, although we investigated the effects of DKK1 on neutrophils, osteoclast-like cells, and macrophages in bone TME, the precise downstream signaling pathways involved in DKK1–CKAP4-mediated immune modulation remain to be explored. Finally, the potential contribution of canonical DKK1 receptors (LRP5/6) to immune regulation within the bone TME was not addressed in this study and whether these classical pathways cooperate with or compensate for CKAP4-dependent signaling warrants further investigation.
Conclusions
In summary, this study highlights the critical impact of myeloid cells in the bone TME and identifies a potential regulatory mechanism underlying immunosuppressive functions. Furthermore, we proposed a novel combination strategy with promising translational potential for bone metastases.
Supporting Information
Conflict of interest statement
No potential conflicts of interest are disclosed.
Author contributions
Conceived and designed the analysis: Jia Wei, Tao Shi.
Collected the data: Yiran Cai, Zizheng Jiang, Ke Zheng.
Contributed data or analysis tools: Jia Wei, Lixia Yu, Baorui Liu.
Performed the analysis: Yuting Luo, Shiji Ren.
Wrote the paper: Yuting Luo, Tao Shi.
Data availability statement
The data generated in this study are available upon request from the corresponding author.
- Received February 25, 2026.
- Accepted June 3, 2026.
- Copyright: © 2026, The Authors
This work is licensed under the Creative Commons Attribution-NonCommercial 4.0 International License.
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